Transaminase biocatalysts

ABSTRACT

The present disclosure relates to polypeptides having transaminase activity, polynucleotides encoding the polypeptides, and methods of using the polypeptides.

1. CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of co-pending U.S. patentapplication Ser. No. 15/915,437, filed Mar. 8, 2018, which is aDivisional of U.S. patent application Ser. No. 15/376,837, filed Dec.13, 2016, now U.S. Pat. No. 9,944,963, which is a Continuation of U.S.patent application Ser. No. 15/140,809, filed Apr. 28, 2016, now U.S.Pat. No. 9,550,982, which is a Divisional of U.S. patent applicationSer. No. 14/818,885, filed Aug. 5, 2015, now U.S. Pat. No. 9,353,355,which is a Divisional of U.S. patent application Ser. No. 14/518,143,filed Oct. 20, 2014, now U.S. Pat. No. 9,133,445, which is a Divisionalof U.S. patent application Ser. No. 13/604,323, filed Sep. 5, 2012, nowU.S. Pat. No. 8,889,380, which is a Continuation of U.S. patentapplication Ser. No. 12/714,397, filed Feb. 26, 2010, now U.S. Pat. No.8,293,507, which claims benefit under 35 U.S.C. § 119(e) of applicationSer. No. 61/155,902, filed Feb. 26, 2009, each of which is incorporatedby reference the contents of which are incorporated herein by referencein its entirety.

2. TECHNICAL FIELD

The present disclosure relates to transaminase biocatalysts and methodsof using the biocatalysts.

3. REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name of 376247-035.txt, creation date of Feb. 26, 2010, with a filesize of 367 Kbytes. The Sequence Listing filed via EFS-Web is part ofthe specification and is herein incorporated by reference.

4. BACKGROUND

Following ingestion of a meal, a group of hormones termed incretins,which include glucagon like peptide-1(GLP-1) and glucose dependentinsulinotropic peptide (GIP), are released. Incretins stimulate insulinrelease and suppress glucagon release in a glucose dependent manner,delay gastric emptying, and increase satiety. Incretins are rapidlydegraded by dipeptidyl peptidase-IV (DPP-4).

Sitagliptin is one of a class of anti-hyperglycemic drugs that inhibitsDPP-4. Inhibiting DPP-4 activity and thereby delaying the inactivationof incretins appears to improve islet function by increasing alpha-celland beta-cell responsiveness to glucose, resulting in improvedglucose-dependent insulin secretion and reduced inappropriate glucagonsecretion. Because of its anti-hyperglycemic effects, sitagliptin hasbeen approved for use in the treatment of Type 2 diabetes in numerouscountries.

The current manufacturing process to produce sitagliptin featuresasymmetric hydrogenation of an unprotected enamine amide (U.S. Pat. No.7,468,459, which issued on Dec. 23, 2008, the contents of which areincorporated by reference in their entirety; Shultz et al., 2007, Acc.Chem. Res. 40:1320-1326). Using a rhodium Josiphos-ligand catalyst inmethanol at 50° C. and 250 psi provides sitagliptin as the free basewith about 97% e.e. Crystallization upgrade of the free base yieldssitagliptin with >99.5% e.e. and 84% yield, and subsequent reaction withphosphoric acid affords sitagliptin phosphate monohydrate, the activepharmaceutical ingredient (“API”) in JANUVIA®, in about 79% overallyield from the enamine amide substrate.

Further improvements in the manufacturing process for sitagliptin aredesirable.

5. SUMMARY

The present disclosure provides polypeptides, polynucleotides encodingthe polypeptides and methods of using the polypeptides for thebiocatalytic conversion of4-oxo-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one(the “ketoamide substrate”) to(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]1-(2,4,5-trifluorophenyl)butan-2-amine(the “product) in presence of an amino group donor. The product, havingthe USAN of sitagliptin, is the active ingredient in JANUVIA®, which hasreceived marketing approval in many countries for the treatment of Type2 diabetes.

While naturally occurring transaminases measured by the inventors do notmeasurably act on the ketoamide substrate, the engineered transaminasesof the present disclosure are capable of carrying out the facileconversion of the ketoamide substrate to the product. Thus, in oneaspect, the present disclosure relates to improved transaminases capableof converting4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]1-(2,4,5-trifluorophenyl)butan-2-one(“the ketoamide substrate”) to(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]1-(2,4,5-trifluorophenyl)butan-2-amine(“the product”) in presence of an amino group donor to levels measurableby an analysis technique, such as HPLC-UV absorbance.

In some embodiments, the improved transaminases of the disclosure arecapable of carrying out the conversion of the ketoamide substrate toproduct with an activity that is least equal to or greater than theactivity of the polypeptide of SEQ ID NO:4. In the embodiments herein,the improved transaminases are capable of forming the product inenantiomeric excess of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more.

In some embodiments, the improved transaminases are capable of carryingout the conversion of the ketoamide substrate to product with at least1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50times, 75 times, 100 times, 150 times, 200 times, 300 times, 400 times,500 times, 1000 times, 1500 times, 2000 times or greater than 2000 timesthe activity of the polypeptide of SEQ ID NO:4 under defined reactionconditions. In some embodiments, the reaction conditions comprise atemperature of 45° C., and a pH of about 8.5.

In some embodiments, the improved transaminase polypeptides are capableof converting the ketoamide substrate4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-oneto the product(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-aminein the presence of an amino group donor with an activity that isimproved over the activity of the transaminase of SEQ ID NO: 2 andcomprises an amino acid sequence that is at least 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or moreidentical to the reference sequence of SEQ ID NO:4, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100 or 102.

In some embodiments, the improved transaminase polypeptide can comprisean amino acid sequence comprising one or more residue differences ascompared to the sequence of SEQ ID NO:2 at the following residuepositions corresponding to: X4; X5; X8; X18; X25; X26; X27; X28; X30;X41; X42; X48; X49; X50; X54; X55; X60; X61; X62; X65; X69; X81; X94;X96; X102; X117; X120; X122; X124; X126; X136; X137; X138; X146; X148;X150; X152; X155; X156; X160; X163; X164; X169; X174; X178; X195; X199;X204; X208; X209; X211; X215; X217; X223; X225; X230; X252; X269; X273;X282, X284; X292; X297; X302; X306; X321; and X329. Guidance for thechoice of various amino acid residues that can be present at thespecified residue positions are provided in the detailed descriptionthat follows.

In some embodiments, the improved transaminase polypeptide comprises anamino acid sequence that includes at least one of the followingfeatures: residue corresponding to X69 is cysteine (C) or a non-polar,polar, or aliphatic residue; residue corresponding to X122 is aconstrained, non-polar or aliphatic residue; residue corresponding toX223 is a constrained residue; and residue corresponding to X284 is anon-polar residue.

In some embodiments, the improved transaminase polypeptide comprises anamino acid sequence that includes at least the following features:residue corresponding to X69 is C or a non-polar, polar, or aliphaticresidue, and/or residue corresponding to X284 is a non-polar residue;residue corresponding to X122 is a constrained, non-polar or aliphaticresidue; and residue corresponding to X223 is a constrained residue.

In some embodiments, the improved transaminase polypeptide comprises anamino acid sequence that includes at least the following features:residue corresponding to X69 is C or a non-polar, polar, or aliphaticresidue; residue corresponding to X122 is a constrained, non-polar oraliphatic residue; and residue corresponding to X223 is a constrainedresidue.

In some embodiments, the improved transaminase polypeptide comprises anamino acid sequence that includes at least the following features:residue corresponding to X122 is a constrained, non-polar or aliphaticresidue; residue corresponding to X223 is a constrained residue; andX284 is a non-polar residue.

In some embodiments, the improved transaminase polypeptide comprises anamino acid sequence that includes at least the following features:residue corresponding to X69 is C or a non-polar, polar or aliphaticresidue; residue corresponding to X122 is a constrained, non-polar oraliphatic residue; residue corresponding to X223 is a constrainedresidue; and residue corresponding to X284 is a non-polar residue.

In some embodiments, the improved transaminase polypeptide comprises anamino acid sequence that includes, in addition to the features describedherein for one or more of residue positions X69, X122, X223, and X284,further includes at least the following features: X26 is an aromatic orconstrained residue, and/or X62 is an aromatic or polar residue; X65 isan aliphatic residue; X136 is an aromatic residue; X199 is an aliphaticor aromatic residue; and X209 is an aliphatic residue.

In some embodiments, the improved transaminase polypeptide comprises anamino acid sequence that includes, in addition to the features describedherein for one or more of residue positions X69, X122, X223, and X284,further includes at least the following features: X61 is an aromaticresidue; X62 is an aromatic or polar residue; X65 is an aliphaticresidue; X94 is an aliphatic residue; X136 is an aromatic residue; X199is an aliphatic or aromatic residue; X209 is an aliphatic residue; X215is a C; and X282 is a polar residue.

In some embodiments, the improved transaminase polypeptide comprises anamino acid sequence that includes, in addition to the features describedherein for one or more of residue positions X69, X122, X223, and X284,further includes at least the following features: X8 is a constrainedresidue; X61 is an aromatic residue; X62 is an aromatic or polarresidue; X65 is an aliphatic residue; X81 is a non-polar or smallresidue; X94 is an aliphatic residue; X136 is an aromatic residue; X199is an aliphatic or aromatic residue; X209 is an aliphatic residue; X215is a C; X217 is a polar residue; X269 is a constrained residue; X282 isa polar residue; X297 is a polar residue; and X321 is a constrainedresidue.

In some embodiments, the improved transaminase polypeptide comprises anamino acid sequence that includes, in addition to the features describedherein for one or more of residue positions X69, X122, X223, and X284,further includes at least the following features: X8 is a constrainedresidue; X60 is an aromatic residue; X61 is an aromatic residue; X62 isan aromatic or polar residue; X65 is an aliphatic residue; X81 is anon-polar residue; X94 is an aliphatic residue; X96 is an aliphaticresidue; X124 is a polar or constrained residue; X136 is an aromaticresidue; X169 is an aliphatic residue; X199 is an aliphatic or aromaticresidue; X209 is an aliphatic residue; X215 is a C; X217 is a polarresidue; X269 is a constrained residue; X273 is an aromatic residue;X282 is a polar residue; X297 is a polar residue; and X321 is aconstrained residue.

In some embodiments, the improved engineered transaminase polypeptidecomprises an amino acid sequence corresponding to the sequence of SEQ IDNO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38,40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166 or 168.

In a further aspect, the present disclosure provides polynucleotidesencoding the improved engineered transaminase polypeptides. In someembodiments, the polynucleotides can be part of an expression vectorhaving one or more control sequences for the expression of thetransaminase polypeptide. In some embodiments, the polynucleotide cancomprise a sequence corresponding to the sequence of SEQ ID NO: 3, 5, 7,9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79,81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111,113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, or 167.

In another aspect, the present disclosure provides host cells comprisingthe polynucleotides encoding the engineered transaminases or expressionvectors capable of expressing the engineered transaminases. In someembodiments, the host cell can be a bacterial host cells, such as E.coli. The host cells can be used for the expression and isolation of theengineered transaminase enzymes described herein, or, alternatively,they can be used directly for the conversion of the ketoamide substrateto product.

In some embodiments, the engineered transaminases, in the form of wholecells, crude extracts, isolated polypeptides, or purified polypeptides,can be used individually or as a combination of different engineeredtransaminases.

In a further aspect, the improved engineered transaminase polypeptidesdescribed herein can be used in a process for transamination of certainamino group acceptors (e.g., a ketone acceptor) in presence of an aminogroup donor. In some embodiments, the transaminases can be used in aprocess for preparing a compound of structural formula (I):

having the indicated stereochemical configuration at the stereogeniccenter marked with an *; in an enantiomeric excess of at least 70% overthe opposite enantiomer, wherein

Z is OR² or NR²R³;

R¹ is C₁₋₈ alkyl, aryl, heteroaryl, aryl-C₁₋₂ alkyl, or heteroaryl-C₁₋₂alkyl;

R² and R³ are each independently hydrogen, C₁₋₈ alkyl, aryl, oraryl-C₁₋₂ alkyl; or

R² and R³ together with the nitrogen atom to which they are attachedform a 4- to 7-membered heterocyclic ring system optionally containingan additional heteroatom selected from O, S, NH, and NC₁₋₄ alkyl, theheterocyclic ring being unsubstituted or substituted with one to threesubstituents independently selected from oxo, hydroxy, halogen, C₁₋₄alkoxy, and C₁₋₄ alkyl, wherein alkyl and alkoxy are unsubstituted orsubstituted with one to five fluorines; and the heterocyclic ring systembeing optionally fused with a 5- to 6-membered saturated or aromaticcarbocyclic ring system or a 5- to 6-membered saturated or aromaticheterocyclic ring system containing one to two heteroatoms selected fromO, S, and NC₀₋₄ alkyl, the fused ring system being unsubstituted orsubstituted with one to two substituents selected from hydroxy, amino,fluorine, C₁₋₄ alkyl, C₁₋₄ alkoxy, and trifluoromethyl, wherein theprocess comprises the step of contacting a prochiral ketone ofstructural formula (II):

with an improved engineered transaminase polypeptide disclosed above inthe presence of an amino group donor in a suitable organic solvent undersuitable reaction conditions for the conversion of the compound offormula (II) to the compound of formula (I).

In some embodiments, the improved engineered transaminase polypeptidesdescribed herein can be used in a process for preparing a compound ofstructural formula (1):

having the (R)-configuration at the stereogenic center marked with an***, in an enantiomeric excess of at least 70% over the enantiomerhaving the opposite (S)-configuration; wherein

Ar is phenyl which is unsubstituted or substituted with one to fivesubstituents independently selected from the group consisting offluorine, trifluoromethyl, and trifluoromethoxy; and

R⁴ is hydrogen or C₁₋₄ alkyl unsubstituted or substituted with one tofive fluorines

wherein, the process comprises the step of contacting a prochiral ketoneof structural formula (2):

with an improved engineered transaminase polypeptide disclosed herein inthe presence of an amino group donor in a suitable organic solvent undersuitable reaction conditions for the conversion of the compound offormula (2) to the compound of formula (1). In some embodiments of theprocess, the Ar of formula (2) is 2,5-difluorophenyl or2,4,5-trifluorophenyl, and R⁴ is trifluoromethyl. In some embodiments ofthe process, the Ar of formula (2) is 2,4,5-trifluorophenyl.

In some embodiments, the improved engineered transaminase polypeptidescan be used in a process for preparing a compound of formula (1a),(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine, inenantiomeric excess:

In these embodiments, the process comprises the step of contacting aprochiral ketone of structural formula (2a),4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]1-(2,4,5-trifluorophenyl)butan-2-one):

with an improved transaminase polypeptide disclosed herein in thepresence of an amino group donor in a suitable organic solvent undersuitable reaction conditions for the conversion of the compound offormula (2a) to the compound of formula (1a).

In some embodiments of the above processes, the compound of formula (I),the compound of formula (1) or the compound of formula (1a) is producedin at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more enantiomeric excess. In some embodiments of the processes, thecompound of formula (I), the compound of formula (1) or the compound offormula (1a) is produced in at least 99% enantiomeric excess.

In some embodiments of the above processes where the choice of the aminogroup donor results in a carbonyl by-product that has a vapor pressurehigher than water (e.g., a low boiling co-product such as a volatileorganic carbonyl compound), the process can be carried out wherein thecarbonyl by-product is removed by sparging the reaction solution with anon-reactive gas (e.g., nitrogen) or by applying a vacuum to lower thereaction pressure and removing the carbonyl by-product present in thegas phase.

The improved engineered transaminase polypeptides useful for the aboveprocesses can comprise an amino acid sequence selected from SEQ ID NO:4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136,138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164,166 or 168.

In a further aspect, the present disclosure provides processes forpreparing(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amineusing the engineered transaminases disclosed herein. In someembodiments, the process comprises contacting a ketoamide substrate4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-onein presence of an amino group donor with an engineered transaminasepolypeptide described herein under reaction conditions suitable forconverting the ketoamide substrate to product(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]1-(2,4,5-trifluorophenyl)butan-2-amine.

In some embodiments, the process is capable of forming the product(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro [1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine in atleast 90% enantiomeric excess.

In some embodiments, the process is capable of forming the product(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine in atleast 99% enantiomeric excess.

In some embodiments, the process for converting ketoamide substrate4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]1-(2,4,5-trifluorophenyl)butan-2-oneto product(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]1-(2,4,5-trifluorophenyl)butan-2-aminecomprises contacting the ketoamide substrate at about 50 g/L with about5 g/L of a transaminase described herein under reaction conditions of pH8.5 and 45° C. in presence of 1 M isopropylamine, wherein at least 90%of the ketoamide substrate is converted to product in 24 hrs. In someembodiments, the transaminase polypeptide capable of carrying out theforegoing reaction comprises an amino acid sequence corresponding to SEQID NO: 80, 86, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168.

6. DETAILED DESCRIPTION

The present disclosure provides highly stereoselective and efficientbiocatalysts capable of mediating transformations involvingtransamination of certain amino group acceptors, e.g., the synthesis ofsitagliptin. The biocatalysts are engineered transaminase polypeptidesthat can convert the substrate of formula (2a),4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]1-(2,4,5-trifluorophenyl)butan-2-one(the “ketoamide substrate”), to the product of formula (1a)(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine(the “product) in presence of an amino group donor of formula (3), asfollows:

In certain embodiments, the engineered transaminases are derived fromthe naturally occurring transaminase of Arthrobacter sp KNK168, which isan R-selective pyridoxal 5′-phosphate dependent enzyme that can catalyzethe reversible transfer of an amino group between an amino group donorand an amino group acceptor, typically a prochiral ketone (see, e.g.,Iwasaki et al., 2006, Appl. Microbiol. Biotechnol. 69: 499-505; and U.S.Pat. No. 7,169,592, each of which is hereby incorporated by referenceherein). The R-stereoselective transamination activity of the naturallyoccurring transaminase from Arthrobacter sp. KNK168 has beendemonstrated on 3,4-dimethoxyphenylacetone, but the naturally occurringenzyme and the transaminase of SEQ ID NO:2 do not display measurableactivity for the ketoamide substrate (2a),4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]1-(2,4,5-trifluorophenyl)butan-2-one.The transaminase of SEQ ID NO:2 differs from the naturally occurringenzyme from Arthrobacter sp. KNK168 in having a substitution ofisoleucine (I) at residue position X306 with valine (V). To overcomethese shortcomings, the transaminase of SEQ ID NO:2 has been engineeredto mediate the efficient conversion of the ketoamide substrate offormula (2a) to the product of formula (1a) in the presence of an aminogroup donor, such as isopropylamine. The conversion can be carried outunder mild conditions with high % conversion and stereoselectivity,making the process applicable to high volume production of sitagliptin.

6.1 ABBREVIATIONS AND DEFINITIONS

For the purposes of the descriptions herein, the abbreviations used forthe genetically encoded amino acids are conventional and are as follows:

Amino Acid Three-Letter Abbreviation One-Letter Abbreviation Alanine AlaA Arginine Arg R Asparagine Asn N Aspartate Asp D Cysteine Cys CGlutamate Glu E Glutamine Gln Q Glycine Gly G Histidine His H IsoleucineIle I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe FProline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine TyrY Valine Val V

When the three-letter abbreviations are used, unless specificallypreceded by an “L” or a “D” or clear from the context in which theabbreviation is used, the amino acid may be in either the L- orD-configuration about α-carbon (Cα). For example, whereas “Ala”designates alanine without specifying the configuration about the αcarbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine,respectively. When the one-letter abbreviations are used, upper caseletters designate amino acids in the L-configuration about the a-carbonand lower case letters designate amino acids in the D-configurationabout the α-carbon. For example, “A” designates L-alanine and “a”designates D-alanine. When peptide sequences are presented as a stringof one-letter or three-letter abbreviations (or mixtures thereof), thesequences are presented in the N→C direction in accordance with commonconvention.

The technical and scientific terms used in the descriptions herein willhave the meanings commonly understood by one of ordinary skill in theart, unless specifically defined otherwise. Accordingly, the followingterms are intended to have the following meanings.

“Aminotransferase” and “transaminase” are used interchangeably herein torefer to a polypeptide having an enzymatic capability of transferring anamino group (NH₂) and a hydrogen atom from a primary amine (3) to anacceptor carbonyl compound (2), converting the amine donor into itscorresponding carbonyl compound (4) and the acceptor into itscorresponding primary amine (1):

In the embodiments herein, the transaminase polypeptides are capable ofenantioselectively converting the compound of formula (2a) to thecompound of formula (1a) in the presence of an amino group donor offormula (3).

“Protein”, “polypeptide,” and “peptide” are used interchangeably hereinto denote a polymer of at least two amino acids covalently linked by anamide bond, regardless of length or post-translational modification(e.g., glycosylation, phosphorylation, lipidation, myristilation,ubiquitination, etc.). Included within this definition are D- andL-amino acids, and mixtures of D- and L-amino acids.

“Substrate” as used herein refers to an amino group acceptor, such as aketone, that accepts the amino group from an amino group donor in areaction mediated by a transaminase. In the context of the presentdisclosure, substrate for the transaminase includes, among others, thecompound of formula (II), the compound of formula (2) and the compoundof formula (2a), as further described herein. A “ketoamide substrate”specifically refers to the compound of formula (2a),4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]1-(2,4,5-trifluorophenyl)butan-2-one.

“Amino group donor” refers to an amino compound which is capable ofdonating an amino group to an acceptor carbonyl compound (i.e., an aminogroup acceptor), thereby becoming a carbonyl by-product Amino groupdonors are molecules of general formula (3),

in which each of R¹, R², when taken independently, is an alkyl, analkylaryl group, or aryl group which is unsubstituted or substitutedwith one or more enzymatically non-inhibiting groups. R¹ can be the sameor different from R² in structure or chirality. The groups R¹ and R²,taken together, may form a ring that is unsubstituted, substituted, orfused to other rings. Typical amino group donors that can be used withthe invention include chiral and achiral amino acids, and chiral andachiral amines

“Chiral amine” refers to amines of general formula R¹—CH(NH₂)—R² whereinR¹ and R² are nonidentical and is employed herein in its broadest sense,including a wide variety of aliphatic and alicyclic compounds ofdifferent, and mixed, functional types, characterized by the presence ofa primary amino group bound to a secondary carbon atom which, inaddition to a hydrogen atom, carries either (i) a divalent group forminga chiral cyclic structure, or (ii) two substituents (other thanhydrogen) differing from each other in structure or chirality. Divalentgroups forming a chiral cyclic structure include, for example,2-methylbutane-1,4-diyl, pentane-1,4-diyl, hexane-1,4-diyl,hexane-1,5-diyl, 2-methylpentane-1,5-diyl. The two differentsubstituents on the secondary carbon atom (R¹ and R² above) also canvary widely and include alkyl, aralkyl, aryl, halo, hydroxy, loweralkyl, lower alkoxy, lower alkylthio, cycloalkyl, carboxy, carboalkoxy,carbamoyl, mono- and di-(lower alkyl) substituted carbamoyl,trifluoromethyl, phenyl, nitro, amino, mono- and di-(lower alkyl)substituted amino, alkylsulfonyl, arylsulfonyl, alkylcarboxamido,arylcarboxamido, etc., as well as alkyl, aralkyl, or aryl substituted bythe foregoing.

“Carbonyl by-product” refers to the carbonyl compound formed from theamino group donor when the amino group on the amino group donor istransferred to the amino group acceptor in a transamination reaction.The carbonyl by-product has the general structure of formula (4):

wherein R¹ and R² are defined above for the amino group donor.

“Pyridoxal-phosphate”, “PLP”, “pyridoxal-5′-phosphate”, “PYP”, and “P5P”are used interchangeably herein to refer to the compound that acts as acoenzyme in transaminase reactions. In some embodiments, pyridoxalphosphate is defined by the structure1-(4′-formyl-3′-hydroxy-2′-methyl-5′-pyridyl)methoxyphosphonic acid, CASnumber [54-47-7]. Pyridoxal-5′-phosphate is produced in vivo byphosphorylation and oxidation of pyridoxol (also known as pyridoxine orVitamin B6). In transamination reactions using transaminase enzymes, theamino group of the amino group donor is transferred to the coenzyme toproduce a keto byproduct, while pyridoxal-5′-phosphate is converted topyridoxamine phosphate. Pyridoxal-5′-phosphate is regenerated byreaction with a different keto compound (the amino group acceptor). Thetransfer of the amino group from pyridoxamine phosphate to the aminoacceptor produces a chiral amine and regenerates the coenzyme. Thepyridoxal-5′-phosphate of the current invention can be replaced by othermembers of the vitamin B6 family, including, among others, pyridoxal(PL), pyridoxamine (PM), and their phosphorylated counterparts;pyridoxine phosphate (PNP), and pyridoxamine phosphate (PMP).

“Coding sequence” refers to that portion of a nucleic acid (e.g., agene) that encodes an amino acid sequence of a protein.

“Naturally occurring” or “wild-type” refers to the form found in nature.For example, a naturally occurring or wild-type polypeptide orpolynucleotide sequence is a sequence present in an organism that can beisolated from a source in nature and which has not been intentionallymodified by human manipulation.

“Recombinant” when used with reference to, e.g., a cell, nucleic acid,or polypeptide, refers to a material, or a material corresponding to thenatural or native form of the material, that has been modified in amanner that would not otherwise exist in nature, or is identical theretobut produced or derived from synthetic materials and/or by manipulationusing recombinant techniques. Non-limiting examples include, amongothers, recombinant cells expressing genes that are not found within thenative (non-recombinant) form of the cell or express native genes thatare otherwise expressed at a different level.

“Percentage of sequence identity,” “percent identity,” and “percentidentical” are used herein to refer to comparisons betweenpolynucleotide sequences or polypeptide sequences, and are determined bycomparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide or polypeptide sequence in thecomparison window may comprise additions or deletions (i.e., gaps) ascompared to the reference sequence for optimal alignment of the twosequences. The percentage is calculated by determining the number ofpositions at which either the identical nucleic acid base or amino acidresidue occurs in both sequences or a nucleic acid base or amino acidresidue is aligned with a gap to yield the number of matched positions,dividing the number of matched positions by the total number ofpositions in the window of comparison and multiplying the result by 100to yield the percentage of sequence identity. Determination of optimalalignment and percent sequence identity is performed using the BLAST andBLAST 2.0 algorithms (see e.g., Altschul et al., 1990, J. Mol. Biol.215: 403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information website.

Briefly, the BLAST analyses involve first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as, the neighborhood word scorethreshold (Altschul et al, supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA89:10915).

Numerous other algorithms are available that function similarly to BLASTin providing percent identity for two sequences. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by thehomology alignment algorithm of Needleman and Wunsch, 1970, J. Mol.Biol. 48:443, by the search for similarity method of Pearson and Lipman,1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe GCG Wisconsin Software Package), or by visual inspection (seegenerally, Current Protocols in Molecular Biology, F. M. Ausubel et al.,eds., Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (1995 Supplement)(Ausubel)). Additionally, determination of sequence alignment andpercent sequence identity can employ the BESTFIT or GAP programs in theGCG Wisconsin Software package (Accelrys, Madison Wis.), using defaultparameters provided.

“Reference sequence” refers to a defined sequence to which another(e.g., altered) sequence is compared. A reference sequence may be asubset of a larger sequence, for example, a segment of a full-lengthgene or polypeptide sequence. Generally, a reference sequence is atleast 20 nucleotide or amino acid residues in length, at least 25residues in length, at least 50 residues in length, or the full lengthof the nucleic acid or polypeptide. Since two polynucleotides orpolypeptides may each (1) comprise a sequence (i.e., a portion of thecomplete sequence) that is similar between the two sequences, and (2)may further comprise a sequence that is divergent between the twosequences, sequence comparisons between two (or more) polynucleotides orpolypeptide are typically performed by comparing sequences of the twopolynucleotides over a comparison window to identify and compare localregions of sequence similarity.

The term “reference sequence” is not intended to be limited to wild-typesequences, and can include engineered or altered sequences. For example,in some embodiments, a “reference sequence” can be a previouslyengineered or altered amino acid sequence. For instance, a “referencesequence based on SEQ ID NO:2 having a glycine residue at position X284”refers to a reference sequence corresponding to SEQ ID NO:2 with aglycine residue at X284 (the un-altered version of SEQ ID NO:2 hasalanine at X284).

“Comparison window” refers to a conceptual segment of at least about 20contiguous nucleotide positions or amino acids residues wherein asequence may be compared to a reference sequence of at least 20contiguous nucleotides or amino acids and wherein the portion of thesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. The comparison window can be longer than 20contiguous residues, and includes, optionally 30, 40, 50, 100, or longerwindows.

“Substantial identity” refers to a polynucleotide or polypeptidesequence that has at least 80 percent sequence identity, at least 85percent sequence identity, at least 89 percent sequence identity, atleast 95 percent sequence identity, and even at least 99 percentsequence identity as compared to a reference sequence over a comparisonwindow of at least 20 residue positions, frequently over a window of atleast 30-50 residues, wherein the percentage of sequence identity iscalculated by comparing the reference sequence to a sequence thatincludes deletions or additions which total 20 percent or less of thereference sequence over the window of comparison. In specificembodiments applied to polypeptides, the term “substantial identity”means that two polypeptide sequences, when optimally aligned, such as bythe programs GAP or BESTFIT using default gap weights, share at least 80percent sequence identity, preferably at least 89 percent sequenceidentity, at least 95 percent sequence identity or more (e.g., 99percent sequence identity). Preferably, residue positions which are notidentical differ by conservative amino acid substitutions.

“Corresponding to”, “reference to” or “relative to” when used in thecontext of the numbering of a given amino acid or polynucleotidesequence refers to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence. In other words, the residuenumber or residue position of a given polymer is designated with respectto the reference sequence rather than by the actual numerical positionof the residue within the given amino acid or polynucleotide sequence.For example, a given amino acid sequence, such as that of an engineeredtransaminase, can be aligned to a reference sequence by introducing gapsto optimize residue matches between the two sequences. In these cases,although the gaps are present, the numbering of the residue in the givenamino acid or polynucleotide sequence is made with respect to thereference sequence to which it has been aligned.

“Stereoselectivity” refers to the preferential formation in a chemicalor enzymatic reaction of one stereoisomer over another.Stereoselectivity can be partial, where the formation of onestereoisomer is favored over the other, or it may be complete where onlyone stereoisomer is formed. When the stereoisomers are enantiomers, thestereoselectivity is referred to as enantioselectivity, the fraction(typically reported as a percentage) of one enantiomer in the sum ofboth. It is commonly alternatively reported in the art (typically as apercentage) as the enantiomeric excess (c.c.) calculated therefromaccording to the formula [major enantiomer—minor enantiomer]/[majorenantiomer +minor enantiomer]. Where the stereoisomers arediastereoisomers, the stereo selectivity is referred to asdiastereoselectivity, the fraction (typically reported as a percentage)of one diastereomer in a mixture of two diastereomers, commonlyalternatively reported as the diastereomeric excess (d.e.). Enantiomericexcess and diastereomeric excess are types of stereomeric excess.

“Highly stereoselective” refers to a chemical or enzymatic reaction thatis capable of converting a substrate (e.g., formula (2a)) to itscorresponding product (e.g., formula (1a)) with at least about 85%stereoisomeric excess.

“Improved enzyme property” refers to any enzyme property made better ormore desirable for a particular purpose as compared to that propertyfound in a reference enzyme. For the engineered transaminasepolypeptides described herein, the comparison is generally made to thewild-type transaminase enzyme, although in some embodiments, thereference transaminase can be another improved engineered transaminase.Enzyme properties for which improvement can be made include, but are notlimited to, enzymatic activity (which can be expressed in terms ofpercent conversion of the substrate in a period of time), thermalstability, solvent stability, pH activity profile, coenzymerequirements, refractoriness to inhibitors (e.g., product inhibition),stereospecificity, and stereo selectivity (includingenantioselectivity).

“Increased enzymatic activity” or “increased activity” refers to animproved property of an engineered enzyme, which can be represented byan increase in specific activity (e.g., product produced/time/weightprotein) or an increase in percent conversion of the substrate to theproduct (e.g., percent conversion of starting amount of substrate toproduct in a specified time period using a specified amount oftransaminase) as compared to a reference enzyme. Exemplary methods todetermine enzyme activity are provided in the Examples. Any propertyrelating to enzyme activity may be affected, including the classicalenzyme properties of K_(m), V_(max) or k_(cat), changes of which canlead to increased enzymatic activity. Improvements in enzyme activitycan be from about 1.5 times the enzymatic activity of the correspondingwild-type or engineered enzyme, to as much as 2 times, 5 times, 10times, 20 times, 25 times, 50 times, 75 times, 100 times, or moreenzymatic activity than the naturally occurring enzyme (e.g., atransaminase) or another engineered enzyme from which the enzymesexhibiting increased activity were derived. In specific embodiments, theengineered transaminase enzymes of the present disclosure exhibitimproved enzymatic activity in the range of 1.5 to 50 times, 1.5 to 100times or greater than that of the parent transaminase enzyme (i.e., thewild-type or engineered transaminase from which they were derived). Itis understood by the skilled artisan that the activity of any enzyme isdiffusion limited such that the catalytic turnover rate cannot exceedthe diffusion rate of the substrate, including any required coenzymes.The theoretical maximum of the diffusion limit is generally about 10⁸ to10⁹ (M⁻¹ s⁻¹). Hence, any improvements in the enzyme activity of thetransaminase will have an upper limit related to the diffusion rate ofthe substrates acted on by the transaminase enzyme. Transaminaseactivity can be measured by any one of standard assays used formeasuring transaminases, such as change in substrate or productconcentration, or change in concentration of the amino group donor.Comparisons of enzyme activities are made using a defined preparation ofenzyme, a defined assay under a set condition, and one or more definedsubstrates, as further described in detail herein. Generally, whenenzymes in cell lysates are compared, the numbers of cells and theamount of protein assayed are determined as well as use of identicalexpression systems and identical host cells to minimize variations inamount of enzyme produced by the host cells and present in the lysates.

“Conversion” refers to the enzymatic transformation of a substrate tothe corresponding product. “Percent conversion” refers to the percent ofthe substrate that is converted to the product within a period of timeunder specified conditions. Thus, for example, the “enzymatic activity”or “activity” of a transaminase polypeptide can be expressed as “percentconversion” of the substrate to the product.

“Thermostable” or “thermal stable” are used interchangeably to refer toa polypeptide that is resistant to inactivation when exposed to a set oftemperature conditions (e.g., 40-80° C.) for a period of time (e.g.,0.5-24 hrs) compared to the untreated enzyme, thus retaining a certainlevel of residual activity (more than 60% to 80% for example) afterexposure to elevated temperatures.

“Solvent stable” refers to a polypeptide that maintains similar activity(more than e.g., 60% to 80%) after exposure to varying concentrations(e.g., 5-99%) of solvent, (e.g., isopropyl alcohol, dimethylsulfoxide,tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene,butylacetate, methyl tert-butylether, acetonitrile, etc.) for a periodof time (e.g., 0.5-24 hrs) compared to the untreated enzyme.

“pH stable” refers to a polypeptide that maintains similar activity(more than e.g., 60% to 80%) after exposure to low or high pH (e.g.,4.5-6 or 8-12) for a period of time (e.g., 0.5-24 hrs) compared to theuntreated enzyme.

“Thermo- and solvent stable” refers to a polypeptide that is boththermostable and solvent stable.

“Derived from” as used herein in the context of engineered enzymesidentifies the originating enzyme, and/or the gene encoding such enzyme,upon which the engineering was based. For example, the engineeredtransaminase enzyme of SEQ ID NO: 4 was obtained by mutating thetransaminase of SEQ ID NO:2. Thus, this engineered transaminase enzymeof SEQ ID NO:4 is “derived from” the polypeptide of SEQ ID NO:2

“Amino acid” or “residue” as used in context of the polypeptidesdisclosed herein refers to the specific monomer at a sequence position(e.g., P8 indicates that the “amino acid” or “residue” at position 8 ofSEQ ID NO: 2 is a proline.)

“Hydrophilic amino acid or residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of less than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophilicamino acids include L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn(N), L-Gln (Q), L-Asp (D), L-Lys (K) and L-Arg (R).

“Acidic amino acid or residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pK value of less than about 6when the amino acid is included in a peptide or polypeptide. Acidicamino acids typically have negatively charged side chains atphysiological pH due to loss of a hydrogen ion. Genetically encodedacidic amino acids include L-Glu (E) and L-Asp (D).

“Basic amino acid or residue” refers to a hydrophilic amino acid orresidue having a side chain exhibiting a pKa value of greater than about6 when the amino acid is included in a peptide or polypeptide. Basicamino acids typically have positively charged side chains atphysiological pH due to association with hydronium ion. Geneticallyencoded basic amino acids include L-Arg (R) and L-Lys (K).

“Polar amino acid or residue” refers to a hydrophilic amino acid orresidue having a side chain that is uncharged at physiological pH, butwhich has at least one bond in which the pair of electrons shared incommon by two atoms is held more closely by one of the atoms.Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q),L-Ser (S) and L-Thr (T).

“Hydrophobic amino acid or residue” refers to an amino acid or residuehaving a side chain exhibiting a hydrophobicity of greater than zeroaccording to the normalized consensus hydrophobicity scale of Eisenberget al., 1984, J. Mol. Biol. 179:125-142. Genetically encoded hydrophobicamino acids include L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu(L), L-Trp (W), L-Met (M), L-Ala (A) and L-Tyr (Y).

“Aromatic amino acid or residue” refers to a hydrophilic or hydrophobicamino acid or residue having a side chain that includes at least onearomatic or heteroaromatic ring. Genetically encoded aromatic aminoacids include L-Phe (F), L-Tyr (Y) and L-Trp (W). Although owing to thepKa of its heteroaromatic nitrogen atom L-His (H) it is sometimesclassified as a basic residue, or as an aromatic residue as its sidechain includes a heteroaromatic ring, herein histidine is classified asa hydrophilic residue or as a “constrained residue” (see below).

“Constrained amino acid or residue” refers to an amino acid or residuethat has a constrained geometry. Herein, constrained residues includeL-Pro (P) and L-His (H). Histidine has a constrained geometry because ithas a relatively small imidazole ring. Proline has a constrainedgeometry because it also has a five membered ring.

“Non-polar amino acid or residue” refers to a hydrophobic amino acid orresidue having a side chain that is uncharged at physiological pH andwhich has bonds in which the pair of electrons shared in common by twoatoms is generally held equally by each of the two atoms (i.e., the sidechain is not polar). Genetically encoded non-polar amino acids includeL-Gly (G), L-Leu (L), L-Val (V), L-Ile (I), L-Met (M) and L-Ala (A).

“Aliphatic amino acid or residue” refers to a hydrophobic amino acid orresidue having an aliphatic hydrocarbon side chain. Genetically encodedaliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L) and L-Ile(I).

“Cysteine” or L-Cys (C) is unusual in that it can form disulfide bridgeswith other L-Cys (C) amino acids or other sulfanyl- orsulfhydryl-containing amino acids. The “cysteine-like residues” includecysteine and other amino acids that contain sulfhydryl moieties that areavailable for formation of disulfide bridges. The ability of L-Cys (C)(and other amino acids with —SH containing side chains) to exist in apeptide in either the reduced free -SH or oxidized disulfide-bridgedform affects whether L-Cys (C) contributes net hydrophobic orhydrophilic character to a peptide. While L-Cys (C) exhibits ahydrophobicity of 0.29 according to the normalized consensus scale ofEisenberg (Eisenberg et al., 1984, supra), it is to be understood thatfor purposes of the present disclosure L-Cys (C) is categorized into itsown unique group.

“Small amino acid or residue” refers to an amino acid or residue havinga side chain that is composed of a total of three or fewer carbon and/orheteroatoms (excluding the 0-carbon and hydrogens). The small aminoacids or residues may be further categorized as aliphatic, non-polar,polar or acidic small amino acids or residues, in accordance with theabove definitions. Genetically-encoded small amino acids include L-Ala(A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T) and L-Asp(D).

“Hydroxyl-containing amino acid or residue” refers to an amino acidcontaining a hydroxyl (—OH) moiety. Genetically-encodedhydroxyl-containing amino acids include L-Ser (S) L-Thr (T) and L-Tyr(Y).

“Amino acid difference” or “residue difference” refers to a change inthe residue at a specified position of a polypeptide sequence whencompared to a reference sequence. For example, a residue difference atposition X8, where the reference sequence has a serine, refers to achange of the residue at position X8 to any residue other than serine.As disclosed herein, an enzyme can include one or more residuedifferences relative to a reference sequence, where multiple residuedifferences typically are indicated by a list of the specified positionswhere changes are made relative to the reference sequence (e.g., “one ormore residue differences as compared to SEQ ID NO:2 at the followingresidue positions: X4; X8; X26; X48; X60; X61; X62; X65; X81; X94; X96;X102; X124; X136; X137; X150; X152; X160; X163; X169; X174; X178; X195;X199; X208; X209; X211; X215; X217; X225; X230; X252; X269; X273; X282;X292; X297; X306; X321; and X329.”).

“Conservative” amino acid substitutions or mutations refer to theinterchangeability of residues having similar side chains, and thustypically involves substitution of the amino acid in the polypeptidewith amino acids within the same or similar defined class of aminoacids. However, as used herein, in some embodiments, conservativemutations do not include substitutions from a hydrophilic tohydrophilic, hydrophobic to hydrophobic, hydroxyl-containing tohydroxyl-containing, or small to small residue, if the conservativemutation can instead be a substitution from an aliphatic to analiphatic, non-polar to non-polar, polar to polar, acidic to acidic,basic to basic, aromatic to aromatic, or constrained to constrainedresidue. Further, as used herein, A, V, L, or I can be conservativelymutated to either another aliphatic residue or to another non-polarresidue. The table below shows exemplary conservative substitutions.

TABLE 1 Residue Possible Conservative Mutations A, L, V, I Otheraliphatic (A, L, V, I) Other non-polar (A, L, V, I, G, M) G, M Othernon-polar (A, L, V, I, G, M) D, E Other acidic (D, E) K, R Other basic(K, R) P, H Other constrained (P, H) N, Q, S, T Other polar Y, W, FOther aromatic (Y, W, F) C None

“Non-conservative substitution” refers to substitution or mutation of anamino acid in the polypeptide with an amino acid with significantlydiffering side chain properties. Non-conservative substitutions may useamino acids between, rather than within, the defined groups listedabove. In one embodiment, a non-conservative mutation affects (a) thestructure of the peptide backbone in the area of the substitution (e.g.,proline for glycine) (b) the charge or hydrophobicity, or (c) the bulkof the side chain.

“Deletion” refers to modification of the polypeptide by removal of oneor more amino acids from the reference polypeptide. Deletions cancomprise removal of 1 or more amino acids, 2 or more amino acids, 5 ormore amino acids, 10 or more amino acids, 15 or more amino acids, or 20or more amino acids, up to 10% of the total number of amino acids, up to20% of the total number of amino acids, or up to 30% of the total numberof amino acids making up the polypeptide while retaining enzymaticactivity and/or retaining the improved properties of an engineeredtransaminase enzyme. Deletions can be directed to the internal portionsand/or terminal portions of the polypeptide. In various embodiments, thedeletion can comprise a continuous segment or can be discontinuous.

“Insertion” refers to modification of the polypeptide by addition of oneor more amino acids to the reference polypeptide. In some embodiments,the improved engineered transaminase enzymes comprise insertions of oneor more amino acids to the naturally occurring transaminase polypeptideas well as insertions of one or more amino acids to other improvedtransaminase polypeptides. Insertions can be in the internal portions ofthe polypeptide, or to the carboxy or amino terminus. Insertions as usedherein include fusion proteins as is known in the art. The insertion canbe a contiguous segment of amino acids or separated by one or more ofthe amino acids in the naturally occurring polypeptide.

“Fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion, but where the remainingamino acid sequence is identical to the corresponding positions in thesequence. Fragments can be at least 14 amino acids long, at least 20amino acids long, at least 50 amino acids long or longer, and up to 70%,80%, 90%, 95%, 98%, and 99% of the full-length transaminase polypeptide,for example the polypeptide of SEQ ID NO:4.

“Isolated polypeptide” refers to a polypeptide which is substantiallyseparated from other contaminants that naturally accompany it, e.g.,protein, lipids, and polynucleotides. The term embraces polypeptideswhich have been removed or purified from their naturally-occurringenvironment or expression system (e.g., host cell or in vitrosynthesis). The improved transaminase enzymes may be present within acell, present in the cellular medium, or prepared in various forms, suchas lysates or isolated preparations. As such, in some embodiments, theimproved transaminase enzyme can be an isolated polypeptide.

“Substantially pure polypeptide” refers to a composition in which thepolypeptide species is the predominant species present (i.e., on a molaror weight basis it is more abundant than any other individualmacromolecular species in the composition), and is generally asubstantially purified composition when the object species comprises atleast about 50 percent of the macromolecular species present by mole or% weight. Generally, a substantially pure transaminase composition willcomprise about 60% or more, about 70% or more, about 80% or more, about90% or more, about 95% or more, and about 98% or more of allmacromolecular species by mole or % weight present in the composition.In some embodiments, the object species is purified to essentialhomogeneity (i.e., contaminant species cannot be detected in thecomposition by conventional detection methods) wherein the compositionconsists essentially of a single macromolecular species. Solventspecies, small molecules (<500 Daltons), and elemental ion species arenot considered macromolecular species. In some embodiments, the isolatedimproved transaminases polypeptide is a substantially pure polypeptidecomposition.

“Stringent hybridization” is used herein to refer to conditions underwhich nucleic acid hybrids are stable. As known to those of skill in theart, the stability of hybrids is reflected in the melting temperature(T_(m)) of the hybrids. In general, the stability of a hybrid is afunction of ion strength, temperature, G/C content, and the presence ofchaotropic agents. The T_(m) values for polynucleotides can becalculated using known methods for predicting melting temperatures (see,e.g., Baldino et al., Methods Enzymology 168:761-777; Bolton et al.,1962, Proc. Natl. Acad. Sci. USA 48:1390; Bresslauer et al., 1986, Proc.Natl. Acad. Sci USA 83:8893-8897; Freier et al., 1986, Proc. Natl. Acad.Sci USA 83:9373-9377; Kierzek et al., Biochemistry 25:7840-7846; Rychliket al., 1990, Nucleic Acids Res 18:6409-6412 (erratum, 1991, NucleicAcids Res 19:698); Sambrook et al., supra); Suggs et al., 1981, InDevelopmental Biology Using Purified Genes (Brown et al., eds.), pp.683-693, Academic Press; and Wetmur, 1991, Crit Rev Biochem Mol Biol26:227-259. All publications incorporate herein by reference). In someembodiments, the polynucleotide encodes the polypeptide disclosed hereinand hybridizes under defined conditions, such as moderately stringent orhighly stringent conditions, to the complement of a sequence encoding anengineered transaminase enzyme of the present disclosure.

“Hybridization stringency” relates to hybridization conditions, such aswashing conditions, in the hybridization of nucleic acids. Generally,hybridization reactions are performed under conditions of lowerstringency, followed by washes of varying but higher stringency. Theterm “moderately stringent hybridization” refers to conditions thatpermit target-DNA to bind a complementary nucleic acid that has about60% identity, preferably about 75% identity, about 85% identity to thetarget DNA; with greater than about 90% identity totarget-polynucleotide. Exemplary moderately stringent conditions areconditions equivalent to hybridization in 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE,0.2% SDS, at 42° C. “High stringency hybridization” refers generally toconditions that are about 10° C. or less from the thermal meltingtemperature T_(m) as determined under the solution condition for adefined polynucleotide sequence. In some embodiments, a high stringencycondition refers to conditions that permit hybridization of only thosenucleic acid sequences that form stable hybrids in 0.018M NaCl at 65° C.(i.e., if a hybrid is not stable in 0.018M NaCl at 65° C., it will notbe stable under high stringency conditions, as contemplated herein).High stringency conditions can be provided, for example, byhybridization in conditions equivalent to 50% formamide, 5× Denhart'ssolution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.1×SSPE,and 0.1% SDS at 65° C. Another high stringency condition is hybridizingin conditions equivalent to hybridizing in 5X SSC containing 0.1% (w:v)SDS at 65° C. and washing in 0.1×SSC containing 0.1% SDS at 65° C. Otherhigh stringency hybridization conditions, as well as moderatelystringent conditions, are described in the references cited above.

“Heterologous” polynucleotide refers to any polynucleotide that isintroduced into a host cell by laboratory techniques, and includespolynucleotides that are removed from a host cell, subjected tolaboratory manipulation, and then reintroduced into a host cell.

“Codon optimized” refers to changes in the codons of the polynucleotideencoding a protein to those preferentially used in a particular organismsuch that the encoded protein is efficiently expressed in the organismof interest. Although the genetic code is degenerate in that most aminoacids are represented by several codons, called “synonyms” or“synonymous” codons, it is well known that codon usage by particularorganisms is nonrandom and biased towards particular codon triplets.This codon usage bias may be higher in reference to a given gene, genesof common function or ancestral origin, highly expressed proteins versuslow copy number proteins, and the aggregate protein coding regions of anorganism's genome. In some embodiments, the polynucleotides encoding thetransaminases enzymes may be codon optimized for optimal production fromthe host organism selected for expression.

“Preferred, optimal, high codon usage bias codons” refersinterchangeably to codons that are used at higher frequency in theprotein coding regions than other codons that code for the same aminoacid. The preferred codons may be determined in relation to codon usagein a single gene, a set of genes of common function or origin, highlyexpressed genes, the codon frequency in the aggregate protein codingregions of the whole organism, codon frequency in the aggregate proteincoding regions of related organisms, or combinations thereof. Codonswhose frequency increases with the level of gene expression aretypically optimal codons for expression. A variety of methods are knownfor determining the codon frequency (e.g., codon usage, relativesynonymous codon usage) and codon preference in specific organisms,including multivariate analysis, for example, using cluster analysis orcorrespondence analysis, and the effective number of codons used in agene (see GCG CodonPreference, Genetics Computer Group WisconsinPackage; CodonW, John Peden, University of Nottingham; McInerney, J. O,1998, Bioinformatics 14:372-73; Stenico et al., 1994, Nucleic Acids Res.222437-46; Wright, F., 1990, Gene 87:23-29). Codon usage tables areavailable for a growing list of organisms (see for example, Wada et al.,1992, Nucleic Acids Res. 20:2111-2118; Nakamura et al., 2000, Nucl.Acids Res. 28:292; Duret, et al., supra; Henaut and Danchin,“Escherichia coli and Salmonella,” 1996, Neidhardt, et al. Eds., ASMPress, Washington D.C., p. 2047-2066. The data source for obtainingcodon usage may rely on any available nucleotide sequence capable ofcoding for a protein. These data sets include nucleic acid sequencesactually known to encode expressed proteins (e.g., complete proteincoding sequences-CDS), expressed sequence tags (EST), or predictedcoding regions of genomic sequences (see for example, Mount, D.,Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Uberbacher, E.C., 1996, Methods Enzymol. 266:259-281; Tiwari et al., 1997, Comput.Appl. Biosci. 13:263-270).

“Control sequence” is defined herein to include all components, whichare necessary or advantageous for the expression of a polynucleotideand/or polypeptide of the present disclosure. Each control sequence maybe native or foreign to the polynucleotide of interest. Such controlsequences include, but are not limited to, a leader, polyadenylationsequence, propeptide sequence, promoter, signal peptide sequence, andtranscription terminator.

“Operably linked” is defined herein as a configuration in which acontrol sequence is appropriately placed (i.e., in a functionalrelationship) at a position relative to a polynucleotide of interestsuch that the control sequence directs or regulates the expression ofthe polynucleotide and/or polypeptide of interest.

“Promoter sequence” is a nucleic acid sequence that is recognized by ahost cell for expression of a polynucleotide of interest, such as acoding sequence. The control sequence may comprise an appropriatepromoter sequence. The promoter sequence contains transcriptionalcontrol sequences, which mediate the expression of a polynucleotide ofinterest. The promoter may be any nucleic acid sequence which showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

6.2 DETAILED DESCRIPTION OF EMBODIMENTS

In the embodiments herein, the engineered transaminases are improved intheir capability of stereoselectively converting ketoamide substrate4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]1-(2,4,5-trifluorophenyl)butan-2-oneto product(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]1-(2,4,5-trifluorophenyl)butan-2-amineas compared to the transaminase of SEQ ID NO:2. Transaminases, includingthose described herein, typically contain a coenzyme, pyridoxalphosphate (PLP), which participates in the transamination reaction. PLPcan be provided by the host cell in which the polypeptide issynthesized, or provided by adding PLP to a solution of the polypeptide.While the transaminase is described with respect to the amino acidsequence, it will be understood by those skilled in the art that theactive polypeptide contains PLP or a suitable analog as a coenzyme.

In some embodiments, the improvement in enzyme activity is with respectto another engineered transaminase, such as the polypeptide of SEQ IDNO:4. The improved activity on the ketoamide substrate can be manifestedby an increase in the amount of substrate converted to product (e.g.,percent conversion) by the engineered enzyme relative to a referenceenzyme (e.g., the wild-type) under defined conditions. The improvedactivity can include an increased rate of product formation resulting inan increase in conversion of ketoamide substrate to product in a definedtime under a defined condition. The increase in activity (e.g.,increased percent conversion and/or conversion rate) may also becharacterized by conversion of substrate to the same amount of productwith a lower amount of enzyme. The amount of product can be assessed bya variety of techniques, for example, separation of the reaction mixture(e.g., by chromatography) and detection of the separated product by UVabsorbance or tandem mass spectroscopy (MS/MS) (see, e.g., Example 4).An exemplary method of UV detection of product uses an incidentwavelength of 210 nm and a path length of 1.0 cm, which has a detectionlimit for sitagliptin of about 5 μg/mL. UV detection of productgenerally follows separation of the reaction mixture by chromatography,particularly HPLC in a reverse phase chromatographic medium, for exampleAgilent Eclipse XDB-C8 column (4.6×150 mm, 5 μm), using an eluent of45:55 10 mM NH₄Ac/McCN at a flow rate of 1.5 ml/min and a columntemperature 40° C. In some embodiments, the UV detection uses anincident wavelength of 268 nm, which has a detection limit similar tothe detection limit at 210 nm.

In some embodiments, the improvement in enzyme activity is equal to orgreater than the activity of the polypeptide of SEQ ID NO:4 underdefined reaction condition, such as provided in Example 6 or 7. Anexemplary defined reaction condition for comparison to the activity ofSEQ ID NO:2 or SEQ ID NO:4 is about 2 g/L ketoamide substrate, about 0.5M isopropylamine, about 22° C., about pH 7.5, about 5% DMSO, about 100μM PLP, and about 20 mg/mL of transaminase polypeptide, as given belowin the description of reaction conditions for the transaminases listedin Table 2. Defined reaction conditions for comparison to certainengineered transaminases are also provided in the description for thetransaminases listed on Table 2, and in the corresponding descriptionsin Examples 7 to 11. In some embodiments, the engineered transaminaseshave at least 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40times, 50 times, 75 times, 100 times, 150 times, 200 times, 300 times,400 times, 500 times, 1000 times, 1500 times, 2000 times or greater than2000 times the activity of the polypeptide of SEQ ID NO:4 under thedefined reaction condition. Given that the transaminase enzyme of SEQ IDNO:2 does not act measurably on the ketoamide substrate4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-]pyrazin-7(8H)-yl]1-(2,4,5-trifluorophenyl)butan-2-one,an engineered transaminase with activity equal to or greater than SEQ IDNO:4 in converting the ketoamide substrate to the corresponding productis improved over the enzyme represented by SEQ ID NO:2.

In some embodiments, the improved enzymatic activity is also associatedwith other improvements in enzyme property. In some embodiments, theimprovement in enzyme property is with respect to thermal stability,such as at 45° C. or higher.

In some embodiments, the improved enzymatic activity is also associatedwith improvements in solvent stability, such as in about 25 to about 40%or about 25 to about 50% dimethylsulfoxide (DMSO). In some embodiments,the improved transaminase is resistance to inactivation by a reactioncomponent, such as the amino group donor. In some embodiments, theengineered transaminases are stable to 1 M or up to 2 M isopropylamine.

In some embodiments, the improved transaminase polypeptide is alsocapable of converting ketoamide substrate4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-oneto product (2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-aminewith at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or greater enantiomeric excess (e.e.).

In some embodiments, the engineered transaminase polypeptides of thepresent disclosure are capable of converting the ketoamide substrate4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-oneto the product(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-aminewith an activity that is equal to or greater than the activity of thepolypeptide of SEQ ID NO:4 in the presence of an amino group donor,particularly isopropylamine, and comprises an amino acid sequence thatis at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the reference sequence of SEQ IDNO:4.

In some embodiments, the engineered transaminase polypeptides of thepresent disclosure are capable of converting the ketoamide substrate4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-oneto the product(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-aminein the presence of an amino group donor, particularly isopropylamine,with an activity that is equal to or greater than the polypeptide of SEQID NO:4 and comprises an amino acid sequence that is at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to a reference sequence listed in Table 2, for example,SEQ ID NO: 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96,98, 100 or 102, as further described below.

In some embodiments, the engineered transaminase polypeptides comprisean amino acid sequence that has one or more residue differences ascompared to a transaminase reference sequence. The residue differencescan be non-conservative substitutions, conservative substitutions, or acombination of non-conservative and conservative substitutions. Withrespect to the residue differences and the descriptions of residuepositions, the transaminases provided herein can be described inreference to the amino acid sequence of the naturally occurringtransaminase of Arthrobacter sp KNK168 or the transaminase of SEQ IDNO:2, or an engineered transaminase, such as the polypeptide of SEQ IDNO:4. For the descriptions herein, the amino acid residue position inthe reference sequence is determined in the transaminase beginning fromthe initiating methionine (M) residue (i.e., M represents residueposition 1), although it will be understood by the skilled artisan thatthis initiating methionine residue may be removed by biologicalprocessing machinery, such as in a host cell or in vitro translationsystem, to generate a mature protein lacking the initiating methionineresidue.

The polypeptide sequence position at which a particular amino acid oramino acid change (“residue difference”) is present is sometimesdescribed herein as “Xn”, or “position n”, where n refers to the residueposition with respect to the reference sequence.

A specific substitution mutation, which is a replacement of the specificresidue in a reference sequence with a different specified residue maybe denoted by the conventional notation “X(number)Y”, where X is thesingle letter identifier of the residue in the reference sequence,“number” is the residue position in the reference sequence, and Y is thesingle letter identifier of the residue substitution in the engineeredsequence.

In some embodiments, the residue differences can occur at one or more ofthe following residue positions: X4; X5; X8; X18; X25; X26; X27; X28;X30; X41; X42; X48; X49; X50; X54; X55; X60; X61; X62; X65; X69; X81;X94; X96; X102; X117; X120; X122; X124; X126; X136; X137; X138; X146;X148; X150; X152; X155; X156; X160; X163; X164; X169; X174; X178; X195;X199; X204; X208; X209; X211; X215; X217; X223; X225; X230; X252; X269;X273; X282; X284; X292; X297; X302; X306; X321; and X329. In someembodiments, the residue differences or combinations thereof, areassociated with the improved enzyme properties. In some embodiments, thetransaminase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences at residue positions other than those specific positionsdenoted by “Xn” listed above. In some embodiments, the number ofdifferences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences atthe other amino acid residue positions. In some embodiments, the residuedifferences at other residue positions comprise substitutions withconservative amino acid residues.

In the embodiments herein, the residue differences as compared to SEQ IDNO:2 at residue positions affecting substrate binding on thetransaminase allows accommodation of the ketoamide substrate ofstructural formula (I), further described below, in particular theketoamide substrate4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]1-(2,4,5-trifluorophenyl)butan-2-one.Without being bound by theory, at least two regions, a first substratebinding region and a second substrate binding region, interact withdifferent structural elements of the ketoamide substrate. The firstbinding region comprises residue positions X62, X136, X137, X195, X199,X208, X209, X223, X225, and X282, while the second binding regioncomprises residue positions X69, X122, and X284. Accordingly, thetransaminase polypeptides herein have one or more residue differences atresidue positions comprising X62, X69, X122, X136, X137, X195, X199,X208, X209, X223, X225, X282, and X284. In some embodiments, thetransaminase polypeptides herein have at least two or more, three ormore, four or more, five or more, or six or more residue differences atthe specified residue positions associated with substrate binding.

In some embodiments, the residue differences as compared to SEQ ID NO:2are at one or more of residue positions forming a first substratebinding region comprised of residue positions X62, X136, X137, X195,X199, X208, X209, X223, X225, and X282. Accordingly, in someembodiments, the engineered transaminase comprises an amino acidsequence that includes at least one residue difference as compared toSEQ ID NO:2 at residue positions X62, X136, X137, X195, X199, X208,X209, X223, X225, and X282.

In some embodiments, the residue differences as compared to SEQ ID NO:2are at one or more of residue positions forming a second substratebinding region comprised of residue positions X69, X122, and X284.Accordingly, in some embodiments, the engineered transaminase comprisesan amino acid sequence that includes at least one residue difference ascompared to SEQ ID NO:2 at residue positions X69, X122, and X284.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes residue differences at the first binding regionin combination with residue differences at the second binding region.Accordingly, in some embodiments, the engineered transaminase comprisesan amino acid sequence that includes one or more residue difference ascompared to SEQ ID NO:2 at residue positions X62, X136, X137, X195,X199, X208, X209, X223, X225, and X282 in combination with one or moreresidue difference as compared to SEQ ID NO:2 at residue positions X69,X122, and X284.

In some embodiments of the engineered transaminases of the disclosure,the amino acid residues at a residue position can be can be defined interms of the amino acid “features” (e.g., type or property of aminoacids) that can appear at that position. Thus, in some embodiments theamino acid residues at the positions specified above can be selectedfrom the following features: X4 is an aromatic residue; X5 is a basicresidue; X8 is a constrained residue; X18 is a cysteine (C) or analiphatic residue; X25 is a polar residue; X26 is an aromatic orconstrained residue; X27 is a polar residue; X28 is a constrainedresidue; X30 is polar or non-polar residue; X41 is a constrained orpolar residue; X42 is non-polar residue; X48 is a polar, acidic,aliphatic or non-polar residue; X49 is a polar residue; X50 is analiphatic residue; X54 is a constrained residue; X55 is an aliphaticresidue; X60 is an aromatic residue; X61 is an aromatic residue; X62 isan aromatic or polar residue; X65 is an aliphatic residue; X69 is acysteine (C) or non-polar, polar, or aliphatic residue; X81 is anon-polar residue; X94 is an aliphatic residue; X96 is an aliphaticresidue; X102 is an aliphatic or basic residue; X117 is a non-polarresidue; X120 is an aromatic residue; X122 is a constrained, non-polaror aliphatic residue; X124 is a polar or constrained residue; X126 is apolar residue; X136 is an aromatic residue; X137 is a polar or aliphaticresidue; X138 is a basic or constrained residue; X146 is a basicresidue; X148 is an aliphatic or aromatic residue; X150 is aromatic,constrained or polar residue; X152 is cysteine (C), non-polar,aliphatic, or polar residue; X155 is non-polar or polar residue; X156 isa polar residue; X160 is an aliphatic residue; X163 is an aliphatic orconstrained residue; X164 is an aliphatic or constrained residue; X169is an aliphatic residue; X174 is an aliphatic residue; X178 is a polarresidue; X195 is an aromatic or polar residue; X199 is an aliphatic oraromatic residue; X204 is an aliphatic residue; X208 is cysteine (C) ora constrained, non-polar, aromatic, polar, or basic residue; X209 is analiphatic residue; X211 is an aliphatic residue; X215 is a cysteine (C);X217 is a polar residue; X223 is a constrained residue; X225 is anaromatic residue; X230 is an aliphatic residue; X252 is an aromatic oraliphatic residue; X269 is a constrained residue; X273 is an aromaticresidue; X282 is a polar residue; X284 is a non-polar residue; X292 is apolar residue; X297 is a polar residue; X302 is an aliphatic residue;X306 is an aliphatic residue; X321 is a constrained residue, and X329 isa constrained or aromatic residue. In some embodiments, where the aminoacid residue at the corresponding residue position of the referencesequence (e.g., SEQ ID NO:2) are encompassed within the category ofamino acids described for the specified position, a different amino acidwithin that amino acid category can be used in light of the guidanceprovided herein.

In some embodiments, the amino acid residue at the residue positionsspecified above can be selected from the following features: X4 is Y, F,or W, particularly Y; X5 is K or R, particularly K; X8 is H or P,particularly P; X18 is C, A, V, or I, particularly C or I; X25 is N, Q,S, or T, particularly Q; X26 is F, W, H or P, particularly H; X27 is N,Q, S, or T, particularly T; X28 is P or H, particularly P; X30 is N, Q,S, T, G, M, A, V, L or I, particularly Q or M; X41 is P, H, N, Q, S, orT, particularly H or S; X42 is G, M, A, V, L or I, particularly G; X48is N, Q, S, T, D, E, G, M, A, V, L, or I, particularly Q, D, V, G, or A;X49 is N, Q, or T, particularly T; X50 is A, V, L or I, particularly L;X54 is P or H; X55 is A, V, or L, particularly V; X60 is F or W,particularly F; X61 is Y, F, or W, particularly Y; X62 is S, T, N, Q, Y,F, or W, particularly T, Y or F; X65 is A, L or I, particularly A; X69is C, G, M, A, L I, S, T, N or Q, particularly G, C, T, A, or S; X81 isG, M, A, V, L, I, particularly G; X94 is A, V, L or I, particularly I orL; X96 is A, V or L, particularly L; X102 is A, V, L, I, K or R,particularly L or K; X117 is G, M, A, V, L or I, particularly G; X120 isY, W, or F, particularly Y; X122 is G, M, A, V, I, L, P or H,particularly M, I, L, V, or H; X124 is T, N, Q, P, or H, particularly T,H or N; X 126 is N, Q, or T, particularly T; X136 is Y, F or W,particularly Y or F; X137 is S, T, N, Q, A, V, L or I, particularly T orI; X138 is K, P or H, particularly K or P; X146 is K or R, particularlyR; X148 is A, V, L I, W, or F, particularly A or F; X150 is F, W, H, P,S, T, N, or Q, particularly F, H, or S; X152 is C, G, M, A, L, I, S, T,N, or Q, particularly G, I, L, S or C; X155 is N, S, T, G, M, A, V, L orI, particularly M, V or T; X156 is N, Q, S, or T, particularly Q; X160is A, V, L or I, particularly L; X163 is P, H, A, V, or L, particularlyH or V; X164 is A, V, L, I, P or H, particularly V or P; X169 is V, L orI, particularly L; X174 is A, V, L or I, particularly A; X178 is S, N,or Q, particularly S; X195 is F, Y, W, S, T, N or Q, particularly F orQ; X199 is A, L, I, Y, F, W, particularly W or I; X204 is A, V, L, or I,particularly A; X208 is H, C, G, K, N, Y, D or S; X209 is V, L or I,particularly L; X211 is A, V, or I, particularly I; X215 is C; X217 isS, T, N or Q, particularly N; X223 is H or P, particularly P; X225 is Wor Y, particularly Y; X230 is A, V, or L, particularly V; X252 is A, V,I, Y, F, or W, particularly F; X269 is H or P, particularly P; X273 isY, F or W, particularly Y; X282 is S, N or Q, particularly S; X284 is G,M, V, L or I, particularly G; X292 is T, N, or Q, particularly T; X297is S, T, N or Q, particularly S; X302 is A, L, or I, particularly A;X306 is A, L or I, particularly L; X321 is H or P, particularly P; andX329 is H, P, Y, F, or W, particularly H.

In some embodiments, the engineered transaminase polypeptide comprisesan amino acid sequence that includes one or more of the followingfeatures: residue corresponding to X69 is cysteine (C) or a non-polar,polar, or aliphatic residue; residue corresponding to X122 is aconstrained, non-polar or aliphatic residue; residue corresponding toX223 is a constrained residue; and residue corresponding to X284 is anon-polar residue.

In some embodiments, the engineered transaminase polypeptide comprisesan amino acid sequence that includes at least the following features:(1) residue corresponding to X69 is C or a non-polar, aliphatic or polarresidue, and/or residue corresponding to X284 is a non-polar residue;(2) residue corresponding to X122 is a constrained, non-polar oraliphatic residue; and (3) residue corresponding to X223 is aconstrained residue.

In some embodiments, the engineered transaminase polypeptide comprisesan amino acid sequence that includes at least the following features:X69 is C or a non-polar, aliphatic or polar residue; X122 is aconstrained, non-polar or aliphatic residue; and X223 is a constrainedresidue.

In some embodiments, the engineered transaminase polypeptide comprisesan amino acid sequence that includes at least the following features:X69 is C, G, M, A, L, I, S, T, N or Q, particularly G, C, T, A, or S;X122 is G, M, A, V, L, I, P or H, particularly M, I, V, L, or H; andX223 is H or P, particularly P.

In some embodiments, the engineered transaminase polypeptide comprisesan amino acid sequence that includes at least the following features:X122 is a constrained, non-polar or aliphatic residue; X223 is aconstrained residue; and X284 is a non-polar residue.

In some embodiments, the engineered transaminase polypeptide comprisesan amino acid sequence that includes at least the following features:X122 is G, M, A, V, L, I, P or H, particularly M, I, V, L or H; X223 isH or P, particularly P; and X284 is G, M, V, L or I, particularly G.

In some embodiments, the engineered transaminase polypeptide comprisesan amino acid sequence that includes at least the following features:X69 is C or a non-polar, polar or aliphatic residue; X122 is aconstrained, non-polar or aliphatic residue; X223 is a constrainedresidue; and X284 is a non-polar residue.

In some embodiments, the engineered transaminase polypeptide comprisesan amino acid sequence that includes at least the following features:X69 is C, G, M, A, L, I, S, T, N or Q, particularly G, C, T, A, or S;X122 is G, M, A, V, L, I, P or H, particularly M, I, V, L, or H; X223 isH or P, particularly P; and X284 is G, M, A, V, L or I, particularly G.

In some embodiments, the engineered transaminase polypeptide comprisesan amino acid sequence that includes at least the following features:X69 is C or T; X122 is M or I; X223 is P; and X284 is G.

In some embodiments, the engineered transaminase polypeptides with oneor more of the specified features or combinations of features at residuepositions X69, X122, X223, and X284, can additionally have one or moreresidue differences as compared to SEQ ID NO:2 at the following residuepositions: X4; X5; X8; X18; X25; X26; X27; X28; X30; X41; X42; X48; X49;X50; X54; X55; X60; X61; X62; X65; X81; X94; X96; X102; X117; X120;X124; X126; X136; X137; X138; X146; X148; X150; X152; X155; X156; X160;X163; X164; X169; X174; X178; X195; X199; X204; X208; X209; X211; X215;X217; X225; X230; X252; X269; X273; X282, X292; X297; X302; X306; X321;and X329. In additions to residue positions X69, X122, X223, and X284,these other residue positions are associated with effects on variousproperties of the transaminase polypeptide, and thus can have residuedifferences as compared to SEQ ID NO:2 to effect desirable changes inenzyme properties.

As noted above, residue positions X62, X136, X137, X195, X199, X208,X209, X225, and X282 along with residue positions X69, X122, X223, andX284, are associated with binding of the substrate to the enzyme, andthus the transaminase polypeptide can have residue differences at theserecited positions as compared to SEQ ID NO:2 to effect desirable changesin enzyme activity.

Residue positions X4, X5, X8, X26, X48, X60, X65, X81, X96, X102, X124,X160, X163, X169, X174, X178, X211, X217, X225, X230, X252, X269, X273,X292, X297, X306, X321, X329 are also associated with additionalincreases in enzyme activity, and thus the transaminase polypeptide canhave residue differences at these recited positions as compared to SEQID NO:2 to effect additional desirable changes in enzyme activity, forexample increase in efficiency of conversion at high substrate loadingconditions.

Residue positions X18, X25, X27, X28, X30, X41, X42, X49, X50, X54, X55,X117, X120, X126, X138, X146, X148, X150, X152, X155, X156, X164, X204,X302 are associated also with increases in thermostability and/orsolvent stability, such as DMSO, and thus the transaminase polypeptidecan have residue differences at these recited positions as compared toSEQ ID NO:2 to effect desirable changes in thermostability and/orsolvent stability.

Residue positions X61, X94, X215 are associated also with the ability tocarry out the reaction at high concentrations of amino donorisopropylamine, and thus the transaminase polypeptide can have residuedifferences at these recited positions as compared to SEQ ID NO:2 toeffect increase in efficiency of conversion at high (e.g., 1-2 M)concentrations of isopropylamine.

It is to be understood that the residue differences from SEQ ID NO:2 atresidue positions associated with the various properties of the enzymescan be used in various combinations to form transaminase polypeptideshaving desirable enzymatic characteristics, for example combination ofincreases in enzyme activity, solvent and temperate stability, andutilization of amino donor. Exemplary combinations are described herein.

In some embodiments, the amino acid residues for the specified residuepositions can be selected according to the descriptions above. Forexample, the amino acid residues can be selected based on the followingfeatures: X4 is an aromatic residue; X5 is a basic residue; X8 is aconstrained residue; X18 is a cysteine (C) or an aliphatic residue; X25is a polar residue; X26 is an aromatic or constrained residue; X27 is apolar residue; X28 is a constrained residue; X30 is a polar or non-polarresidue; X41 is a constrained or polar residue; X42 is a non-polarresidue; X48 is a polar, acidic, aliphatic or non-polar residue; X49 isa polar residue; X50 is an aliphatic residue; X54 is a constrainedresidue; X55 is an aliphatic residue; X60 is an aromatic residue; X61 isan aromatic residue; X62 is an aromatic or polar residue; X65 is analiphatic residue; X81 is a non-polar residue; X94 is an aliphaticresidue; X96 is an aliphatic residue; X102 is an aliphatic or basicresidue; X117 is a non-polar residue; X120 is an aromatic residue; X124is a polar or constrained residue; X126 is a polar residue; X136 is anaromatic residue; X137 is a polar or aliphatic residue; X138 is a basicor constrained residue; X146 is a basic residue; X148 is an aliphatic oraromatic residue; X150 is an aromatic, constrained or polar residue;X152 is a cysteine (C), non-polar, aliphatic, or polar residue; X155 isa non-polar or polar residue; X156 is a polar residue; X160 is analiphatic residue; X163 is an aliphatic or constrained residue; X164 isan aliphatic or constrained residue; X169 is an aliphatic residue; X174is an aliphatic residue; X178 is a polar residue; X195 is an aromatic orpolar residue; X199 is an aliphatic or aromatic residue; X204 is analiphatic residue; X208 is a cysteine (C) or a constrained, non-polar,aromatic, polar, or basic residue; X209 is an aliphatic residue; X211 isan aliphatic residue; X215 is C; X217 is a polar residue; X225 is anaromatic residue; X230 is an aliphatic residue; X252 is an aromatic oraliphatic residue; X269 is a constrained residue; X273 is an aromaticresidue; X282 is a polar residue; X292 is a polar residue; X297 is apolar residue; X302 is an aliphatic residue; X306 is an aliphaticresidue; X321 is a constrained residue; and X329 is a constrained oraromatic residue. Specific amino acid residues that can be used at theseresidue positions are described above.

In some embodiments, the engineered transaminase having the features atone or more residue positions X69, X122, X223, and X284 as describedabove, can have additionally one or more of the following features: X26is an aromatic or constrained residue; X61 is an aromatic residue; X62is an aromatic or polar residue; X65 is an aliphatic residue; X94 is analiphatic residue; X136 is an aromatic residue; X137 is a polar oraliphatic residue; X199 is an aliphatic or aromatic residue; X209 is analiphatic residue; X215 is C; and X282 is a polar residue.

In some embodiments, in addition to the preceding features, thetransaminase amino acid sequence can include additionally one or more ofthe following features: X8 is a constrained residue; X60 is an aromaticresidue; X81 is a non-polar or small residue; X96 is an aliphaticresidue; X124 is a polar or constrained residue; X169 is an aliphaticresidue; X217 is a polar residue; X269 is a constrained residue; X273 isan aromatic residue; X297 is a polar residue; and X321 is a constrainedresidue.

In some embodiments, in addition to the preceding features, thetransaminase amino acid sequence can include additionally one or more ofthe following features: X4 is an aromatic residue; X48 is a polar,acidic, aliphatic or non-polar residue; X102 is an aliphatic or basicresidue; X150 is aromatic, constrained or polar residue; X152 is C or anon-polar, aliphatic or polar residue; X160 is an aliphatic residue;X163 is an aliphatic or constrained residue; X174 is an aliphaticresidue; X178 is a polar residue; X195 is an aromatic or polar residue;X208 is C or a constrained, non-polar, aromatic, polar, or basicresidue; X211 is an aliphatic residue; X225 is an aromatic residue; X230is an aliphatic residue; X252 is an aromatic or aliphatic residue; X292is a polar residue; X306 is an aliphatic residue; and X329 is aconstrained or aromatic residue.

In some embodiments, the engineered transaminase having the features atone or more or combinations of features at residue positions X69, X122,X223, and X284 as described above, includes at least the followingadditional features: X26 is an aromatic or constrained residue, and/orX62 is an aromatic or polar residue; X65 is an aliphatic residue; X136is an aromatic residue; X199 is an aliphatic or aromatic residue; andX209 is an aliphatic residue.

In some embodiments, the engineered transaminase having the features atone or more residue positions X69, X122, X223, and X284 as describedabove, includes at least the following additional features: X61 is anaromatic residue; X62 is an aromatic or polar residue; X65 is analiphatic residue; X94 is an aliphatic residue; X136 is an aromaticresidue; X199 is an aliphatic or aromatic residue; X209 is an aliphaticresidue; X215 is C, and X282 is a polar residue.

In some embodiments, the engineered transaminase having the features atone or more residue positions X69, X122, X223, and X284 as describedabove, includes at least the following additional features: X8 is aconstrained residue; X61 is an aromatic residue; X62 is an aromatic orpolar residue; X65 is an aliphatic residue; X81 is a non-polar or smallresidue; X94 is an aliphatic residue; X136 is an aromatic residue; X199is an aliphatic or aromatic residue; X209 is an aliphatic residue; X215is a C; X217 is a polar residue; X269 is a constrained residue; X282 isa polar residue X297 is a polar residue; and X321 is a constrainedresidue.

In some embodiments, the engineered transaminase having the features atone or more residue positions X69, X122, X223, and X284 as describedabove, includes at least the following additional features: X8 is aconstrained residue; X60 is an aromatic residue; X61 is an aromaticresidue; X62 is an aromatic or polar residue; X65 is an aliphaticresidue; X81 is a non-polar residue; X94 is an aliphatic residue; X96 isan aliphatic residue; X124 is a polar or constrained residue; X136 is anaromatic residue; X169 is an aliphatic residue; X199 is an aliphatic oraromatic residue; X209 is an aliphatic residue; X215 is C; X217 is apolar residue; X269 is a constrained residue; X273 is an aromaticresidue. X282 is a polar residue; X297 is a polar residue; and X321 is aconstrained residue.

In some embodiments, the engineered transaminase having the features atone or more residue positions X69, X122, X223, and X284 as describedabove, includes at least the following additional features: X8 is aconstrained residue; X60 is an aromatic residue; X61 is an aromaticresidue; X62 is an aromatic or polar residue; X65 is an aliphaticresidue; X81 is a non-polar residue; X94 is an aliphatic residue; X96 isan aliphatic residue; X124 is a polar or constrained residue; X126 is apolar residue; X136 is an aromatic residue; X150 is an aromatic,constrained or polar residue; X152 is a cysteine (C), non-polar,aliphatic, or polar residue; X169 is an aliphatic residue; X199 is analiphatic or aromatic residue; X209 is an aliphatic residue; X215 is C;X217 is a polar residue; X269 is a constrained residue; X273 is anaromatic residue. X282 is a polar residue; X297 is a polar residue; andX321 is a constrained residue.

In some embodiments, the engineered transaminase having the features atone or more residue positions X69, X122, X223, and X284 as describedabove, includes at least the following additional features: X26 is P, H,F, or W, particularly H, and/or X62 is S, T, N, Q, Y, F, or W,particularly T or F; X65 is A, L or I, particularly A; X136 is Y, F orW, particularly Y or F; X199 is A, L, I, Y, F, or W, particularly W orI; and X209 is V, L or I, particularly L.

In some embodiments, the engineered transaminase having the features atone or more residue positions X69, X122, X223, and X284 as describedabove, includes at least the following additional features: X61 is Y, F,or W, particularly Y; X62 is S, T, N, Q, Y, F, or W, particularly T orF; X65 is A, L or I, particularly A; X94 is A, V, L or I, particularly Ior L; X136 is Y, F, or W, particularly Y or F; X199 is A, L, I, Y, F, orW, particularly W or I; X209 is V, L or I, particularly L; X215 is C;and X282 is S, N or Q, particularly S.

In some embodiments, the engineered transaminase having the features atone or more residue positions X69, X122, X223, and X284 as describedabove, includes at least the following additional features: X8 is H orP, particularly P; X61 is Y, F, or W, particularly Y; X62 is S, T, N Q,Y, F, or W, particularly T or F; X65 is A, L or I, particularly A; X81is G, M, A, V, L or I, particularly G; X94 is A, V, L or I, particularlyI or L; X136 is Y, F, or W, particularly Y or F; X199 is A, L, I, Y, F,or W, particularly W or I; X209 is V, L or I, particularly L; X215 is C;X217 is S, T, N, or Q, particularly N; X269 is H or P, particularly P;X282 is S, N or Q, particularly S. X297 is S, T, N or Q, particularly S;and X321 is H or P, particularly P.

In some embodiments, the engineered transaminase having the features atone or more residue positions X69, X122, X223, and X284 as describedabove, includes at least the following additional features: X8 is H orP, particularly P; X60 is F or W, particularly F; X61 is Y, F, or W,particularly Y; X62 is Y, F, W, S, T, N or Q, particularly T or F; X65is A, L or I, particularly A; X81 is G, M, A, V, L or I, particularly G;X94 is A, V, L or I, particularly I or L; X96 is A, V or L, particularlyL; X124 is P, H, T, N, or Q, particularly T, H or N; X136 is Y, F or W,particularly Y or F; X169 is V, L, or I, particularly L; X199 is Y, F,W, A, L or I, particularly W or I; X209 is V, L or I, particularly L;X215 is C; X217 is S, T, N or Q, particularly N; X269 is H or P,particularly P; X273 is Y, F, or W, particularly Y; X282 is S, N or Q,particularly S; X297 is S, T, N or Q, particularly S; and X321 is H orP, particularly P.

In some embodiments, the engineered transaminase having the features atone or more residue positions X69, X122, X223, and X284 as describedabove, includes at least the following additional features: X8 is H orP, particularly P; X60 is F or W, particularly F; X61 is Y, F, or W,particularly Y; X62 is Y, F, W, S, T, N or Q, particularly T or F; X65is A, L or I, particularly A; X81 is G, M, A, V, L or I, particularly G;X94 is A, V, L or I, particularly I or L; X96 is A, V or L, particularlyL; X124 is P, H, T, N, or Q, particularly T, H or N; X 126 is N, Q, orT, particularly T; X136 is Y, F or W, particularly Y or F; X150 is F, W,H, P, S, T, N, or Q, particularly F, H, or S; X152 is C, G, M, A, L, I,S, T, N, or Q, particularly G, I, L, S or C; X169 is V, L, or I,particularly L; X199 is Y, F, W, A, L or I, particularly W or I; X209 isV, L or I, particularly L; X215 is C; X217 is S, T, N or Q, particularlyN; X269 is H or P, particularly P; X273 is Y, F, or W, particularly Y;X282 is S, N or Q, particularly S; X297 is S, T, N or Q, particularly S;and X321 is H or P, particularly P.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes at least the following features: X122 is aconstrained, non-polar or aliphatic residue, particularly M, I, L, V, orH; X223 is a constrained residue, particularly P; X284 is a non-polarresidue, particularly G. In some embodiments, the transaminasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residue differences at otherresidue positions. In some embodiments, the number of differences can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26,30, 35, 40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference amino acid sequence based on SEQ ID NO:2 havingthe features described for the preceding specified residue positions(i.e. X122; X223; and X284) (e.g., SEQ ID NO:8 or 10), with the provisothat the engineered transaminase polypeptide comprises polypeptidecomprises an amino acid sequence that includes at least the featuresdescribed for the specified residues.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes at least the following features: X69 is C or anon-polar, aliphatic or polar residue, particularly G, C, T, A, or S;X122 is a constrained, non-polar or aliphatic residue, particularly M,I, L, V, or H; X223 is a constrained residue, particularly P; and X284is a non-polar residue, particularly G. In some embodiments, thetransaminase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences at other residue positions. In some embodiments, the numberof differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences atthe other residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence based on SEQ IDNO:2 having the features described for preceding specified residuepositions (i.e., X69; X122; X223; and X284)(e.g., SEQ ID NO:4), with theproviso that the engineered transaminase polypeptide comprisespolypeptide comprises an amino acid sequence having at least thefeatures described for the specified residues.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes at least the following features: X65 is analiphatic residue, particularly A; X69 is C or a non-polar, aliphatic orpolar residue, particularly G, C, T, A, or S; X122 is a constrained,non-polar or aliphatic residue, particularly M, I, L, V, or H; and X223is a constrained residue, particularly P. In some embodiments, thetransaminase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences at other residue positions. In some embodiments, the numberof differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences atthe other residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence based on SEQ IDNO:2 having the features described for the preceding specified residuepositions (e.g., SEQ ID NO:6), with the proviso that the engineeredtransaminase polypeptide comprises polypeptide comprises an amino acidsequence that includes at least the features described for the specifiedresidues. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence of SEQ ID NO:6.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes at least the following features: X122 is aconstrained, non-polar or aliphatic residue, particularly M, I, L, V, orH; X174 is an aliphatic residue, particularly A; X223 is a constrainedresidue, particularly P; and X284 is a non-polar residue, particularlyG. In some embodiments, the transaminase polypeptides can haveadditionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45,1-50, 1-55, or 1-60 residue differences at other residue positions. Insome embodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50,55, or 60 residue differences at the other residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence based on SEQ ID NO:2 having the features describedfor preceding specified residue positions (e.g., SEQ ID NO:12), with theproviso that the engineered transaminase polypeptide comprisespolypeptide comprises an amino acid sequence that includes at least thefeatures described for the specified residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:12.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes the following features: In some embodiments, theengineered transaminases comprise an amino acid sequence that includesat least the following features: X26 is an aromatic or constrainedresidue, particularly H; X65 is an aliphatic residue, particularly A;X69 is C or a non-polar, aliphatic or polar residue, particularly G, C,T, A, or S; X122 is a constrained, non-polar or aliphatic residue,particularly M, I, L, V, or H; X223 is a constrained residue,particularly P; and X284 is a non-polar residue, particularly G. In someembodiments, the transaminase polypeptides can have additionally 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or1-60 residue differences at other residue positions. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or60 residue differences at the other residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence based on SEQ ID NO:2 having the features describedfor preceding specified residue positions (e.g., SEQ ID NO:14), with theproviso that the engineered transaminase polypeptide comprisespolypeptide comprises an amino acid sequence that includes at least thefeatures described for the specified residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:14.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes the following features: In some embodiments, theengineered transaminases comprise an amino acid sequence that includesat least the following features: X26 is an aromatic or constrainedresidue, particularly H; X62 is an aromatic or polar residue,particularly T, Y or F; X65 is an aliphatic residue, particularly A; X69is C or a non-polar, aliphatic or polar residue, particularly G, C, T,A, or S; X122 is a constrained, non-polar or aliphatic residue,particularly M, I, L, V, or H; X178 is a polar residue, particularly S;X199 is an aliphatic or aromatic residue, particularly W or I,particularly X223 is a constrained residue, particularly P; X225 is anaromatic residue, particularly Y, X282 is a polar residue, particularlyS; and X284 is a non-polar residue, particularly G. In some embodiments,the transaminase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5,1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20,1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences at other residue positions. In some embodiments, the numberof differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences atthe other residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence based on SEQ IDNO:2 having the features described for preceding specified residuepositions (e.g., SEQ ID NO:16), with the proviso that the engineeredtransaminase polypeptide comprises polypeptide comprises an amino acidsequence that includes at least the features described for the specifiedresidue positions. In some embodiments, the engineered transaminasepolypeptide can comprise an amino acid sequence that is at least about80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% identical to a reference sequence of SEQ ID NO:16.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes the following features: In some embodiments, theengineered transaminases comprise an amino acid sequence that includesat least the following features: X26 is an aromatic or constrainedresidue, particularly H; X62 is an aromatic or polar residue,particularly T, Y or F; X65 is an aliphatic residue, particularly A; X69is C or a non-polar, aliphatic or polar residue, particularly G, C, T,A, or S; X122 is a constrained, non-polar or aliphatic residue,particularly M, I, L, V, or H; X136 is an aromatic residue, particularlyY or F; X199 is an aliphatic or aromatic residue, particularly W or I;X209 is an aliphatic residue, particularly L; X223 is a constrainedresidue, particularly P; X225 is an aromatic residue, particularly Y,X282 is a polar residue, particularly S; and X284 is a non-polarresidue, particularly G. In some embodiments, the transaminasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residue differences at otherresidue positions. In some embodiments, the number of differences can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26,30, 35, 40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO:18), with the proviso that the engineered transaminase polypeptidecomprises polypeptide comprises an amino acid sequence that includes atleast the features described for the specified residue positions. Insome embodiments, the engineered transaminase polypeptide can comprisean amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:18.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes the following features: In some embodiments, theengineered transaminases comprise an amino acid sequence that includesat least the following features: X26 is an aromatic or constrainedresidue, particularly H; X62 is an aromatic or polar residue,particularly T, Y or F; X65 is an aliphatic residue, particularly A; X69is C or a non-polar, aliphatic or polar residue, particularly G, C, T,A, or S; X122 is a constrained, non-polar or aliphatic residue,particularly M, I, L, V, or H; X136 is an aromatic residue, particularlyY or F; X137 is a polar or aliphatic residue, particularly T or I; X199is an aliphatic or aromatic residue, particularly W or I; X209 is analiphatic residue, particularly L; X223 is a constrained residue,particularly P; X282 is a polar residue, particularly S; and X284 is anon-polar residue, particularly G. In some embodiments, the transaminasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residue differences at otherresidue positions. In some embodiments, the number of differences can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26,30, 35, 40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO:20, 22, 28, 30, 32, 34, 38 or 40), with the proviso that theengineered transaminase polypeptide comprises polypeptide comprises anamino acid sequence that includes at least the features described forthe specified residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence of SEQ ID NO:20,22, 28, 30, 32, 34, 38 or 40.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes the following features: In some embodiments, theengineered transaminases comprise an amino acid sequence that includesat least the following features: X26 is an aromatic or constrainedresidue, particularly H; X62 is an aromatic or polar residue,particularly T, Y or F; X65 is an aliphatic residue, particularly A; X69is C or a non-polar, aliphatic or polar residue, particularly G, C, T,A, or S; X122 is a constrained, non-polar or aliphatic residue,particularly M, I, L, V, or H; X136 is an aromatic residue, particularlyY or F; X137 is a polar or aliphatic residue, particularly T or I; X199is an aliphatic or aromatic residue, particularly W or I; X209 is analiphatic residue, particularly L; X223 is a constrained residue,particularly P; X225 is an aromatic residue, particularly Y, X282 is apolar residue, particularly S; and X284 is a non-polar residue,particularly G. In some embodiments, the transaminase polypeptides canhave additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11,1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40,1-45, 1-50, 1-55, or 1-60 residue differences at other residuepositions. In some embodiments, the number of differences can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35,40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO:24), with the proviso that the engineered transaminase polypeptidecomprises polypeptide comprises an amino acid sequence that includes atleast the features described for the specified residue positions. Insome embodiments, the engineered transaminase polypeptide can comprisean amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:24.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes the following features: In some embodiments, theengineered transaminases comprise an amino acid sequence that includesat least the following features: X26 is an aromatic or constrainedresidue, particularly H; X65 is an aliphatic residue, particularly A;X69 is C or a non-polar, aliphatic or polar residue, particularly G, C,T, A, or S; X122 is a constrained, non-polar or aliphatic residue,particularly M, I, L, V, or H; X136 is an aromatic residue, particularlyY or F; X137 is a polar or aliphatic residue, particularly T or I; X174is an aliphatic residue, particularly A; X199 is an aliphatic oraromatic residue, particularly W or I; X209 is an aliphatic residue,particularly L; X223 is a constrained residue, particularly P; X230 isan aliphatic residue, particularly V; and X284 is a non-polar residue,particularly G. In some embodiments, the transaminase polypeptides canhave additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11,1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40,1-45, 1-50, 1-55, or 1-60 residue differences at other residuepositions. In some embodiments, the number of differences can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35,40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO:26), with the proviso that the engineered transaminase polypeptidecomprises polypeptide comprises an amino acid sequence that includes atleast the features described for the specified residue positions. Insome embodiments, the engineered transaminase polypeptide can comprisean amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:26.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes the following features: In some embodiments, theengineered transaminases comprise an amino acid sequence that includesat least the following features: X26 is an aromatic or constrainedresidue, particularly H; X61 is an aromatic residue, particularly Y; X62is an aromatic or polar residue, particularly T, Y or F; X65 is analiphatic residue, particularly A; X69 is C or a non-polar, aliphatic orpolar residue, particularly G, C, T, A, or S; X122 is a constrained,non-polar or aliphatic residue, particularly M, I, L, V, or H; X136 isan aromatic residue, particularly Y or F; X137 is a polar or aliphaticresidue, particularly T or I; X199 is an aliphatic or aromatic residue,particularly W or I; X209 is an aliphatic residue, particularly L; X223is a constrained residue, particularly P; X282 is a polar residue,particularly S; and X284 is a non-polar residue, particularly G. In someembodiments, the transaminase polypeptides can have additionally 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or1-60 residue differences at other residue positions. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or60 residue differences at the other residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence based on SEQ ID NO:2 having the features describedfor preceding specified residue positions (e.g., SEQ ID NO:36), with theproviso that the engineered transaminase polypeptide comprisespolypeptide comprises an amino acid sequence that includes at least thefeatures described for the specified residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:36.

In some embodiments, the engineered transaminases comprise an amino acidsequence that includes the following features: In some embodiments, theengineered transaminases comprise an amino acid sequence that includesat least the following features: X4 is an aromatic residue, particularlyY; X26 is an aromatic or constrained residue, particularly H; X62 is anaromatic or polar residue, particularly T, Y or F; X65 is an aliphaticresidue, particularly A; X69 is C or a non-polar, aliphatic or polarresidue, particularly G, C, T, A, or S; X94 is an aliphatic residue,particularly I or L; X122 is a constrained, non-polar or aliphaticresidue, particularly M, I, L, V, or H; X136 is an aromatic residue,particularly Y or F; X137 is a polar or aliphatic residue, particularlyT or I; X199 is an aliphatic or aromatic residue, particularly W or I;X209 is an aliphatic residue, particularly L; X215 is C; X223 is aconstrained residue, particularly P; X282 is a polar residue,particularly S; and X284 is a non-polar residue, particularly G. In someembodiments, the transaminase polypeptides can have additionally 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or1-60 residue differences at other residue positions. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or60 residue differences at the other residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence based on SEQ ID NO:2 having the features describedfor preceding specified residue positions (e.g., SEQ ID NO:42), with theproviso that the engineered transaminase polypeptide comprisespolypeptide comprises an amino acid sequence that includes at least thefeatures described for the specified residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:42.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X62 is an aromatic orpolar residue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X94 is an aliphatic residue, particularlyI or L; X122 is a constrained, non-polar or aliphatic residue,particularly M, I, L, V, or H; X136 is an aromatic residue, particularlyY or F; X137 is a polar or aliphatic residue, particularly T or I; X199is an aliphatic or aromatic residue, particularly W or I; X209 is analiphatic residue, particularly L; X215 is a C; X223 is a constrainedresidue, particularly P; X282 is a polar residue, particularly S; andX284 is a non-polar residue, particularly G. In some embodiments, thetransaminase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences at other residue positions. In some embodiments, the numberof differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences atthe other residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence based on SEQ IDNO:2 having the features described for preceding specified residuepositions (e.g., SEQ ID NO: 44, 46, or 48), with the proviso that theengineered transaminase polypeptide comprises polypeptide comprises anamino acid sequence that includes at least the features described forthe specified residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence of SEQ ID NO:44,46, or 48.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly P; X62 is an aromatic or polar residue,particularly T, Y or F; X65 is an aliphatic residue, particularly A; X69is C or a non-polar, aliphatic or polar residue, particularly G, C, T,A, or S; X94 is an aliphatic residue, particularly I or L; X122 is aconstrained, non-polar or aliphatic residue, particularly M, I, L, V, orH; X136 is an aromatic residue, particularly Y or F; X137 is a polar oraliphatic residue, particularly T or I; X199 is an aliphatic or aromaticresidue, particularly W or I; X209 is an aliphatic residue, particularlyL; X215 is a cysteine (C); X223 is a constrained residue, particularlyP; X282 is a polar residue, particularly S; and X284 is a non-polarresidue, particularly G. In some embodiments, the transaminasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residue differences at otherresidue positions. In some embodiments, the number of differences can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26,30, 35, 40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO: 50), with the proviso that the engineered transaminasepolypeptide comprises polypeptide comprises an amino acid sequence thatincludes at least the features described for the specified residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence of SEQ ID NO:50.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X61 is an aromaticresidue, particularly Y; X62 is an aromatic or polar residue,particularly T, Y or F; X65 is an aliphatic residue, particularly A; X69is C or a non-polar, aliphatic or polar residue, particularly G, C, T,A, or S; X94 is an aliphatic residue, particularly I or L; X122 is aconstrained, non-polar or aliphatic residue, particularly M, I, L, V, orH; X136 is an aromatic residue, particularly Y or F; X137 is a polar oraliphatic residue, particularly T or I; X152 is C, non-polar, aliphatic,or polar residue, particularly G, I, L, S or C; X199 is an aliphatic oraromatic residue, particularly W or I; X209 is an aliphatic residue,particularly L; X215 is a C; X223 is a constrained residue, particularlyP; X282 is a polar residue, particularly S; and X284 is a non-polarresidue, particularly G. In some embodiments, the transaminasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residue differences at otherresidue positions. In some embodiments, the number of differences can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26,30, 35, 40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO: 52), with the proviso that the engineered transaminasepolypeptide comprises polypeptide comprises an amino acid sequence thatincludes at least the features described for the specified residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence of SEQ ID NO:52.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X61 is an aromaticresidue, particularly Y; X62 is an aromatic or polar residue,particularly T, Y or F; X65 is an aliphatic residue, particularly A; X69is C or a non-polar, aliphatic or polar residue, particularly G, C, T,A, or S; X94 is an aliphatic residue, particularly I or L; X122 is aconstrained, non-polar or aliphatic residue, particularly M, I, L, V, orH; X136 is an aromatic residue, particularly Y or F; X137 is a polar oraliphatic residue, particularly T or I; X199 is an aliphatic or aromaticresidue, particularly W or I; X209 is an aliphatic residue, particularlyL; X215 is a C; X223 is a constrained residue, particularly P; X282 is apolar residue, particularly S; and X284 is a non-polar residue,particularly G. In some embodiments, the transaminase polypeptides canhave additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11,1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40,1-45, 1-50, 1-55, or 1-60 residue differences at other residuepositions. In some embodiments, the number of differences can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35,40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO: 54 or 56), with the proviso that the engineered transaminasepolypeptide comprises polypeptide comprises an amino acid sequence thatincludes at least the features described for the specified residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence of SEQ ID NO:54 or 56.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X61 is an aromaticresidue, particularly Y; X62 is an aromatic or polar residue,particularly T, Y or F; X65 is an aliphatic residue, particularly A; X69is C or a non-polar, aliphatic or polar residue, particularly G, C, T,A, or S; X94 is an aliphatic residue, particularly I or L; X122 is aconstrained, non-polar or aliphatic residue, particularly M, I, L, V, orH; X136 is an aromatic residue, particularly Y or F; X199 is analiphatic or aromatic residue, particularly W or I; X209 is an aliphaticresidue, particularly L; X215 is a C; X223 is a constrained residue,particularly P; X282 is a polar residue, particularly S; and X284 is anon-polar residue, particularly G. In some embodiments, the transaminasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residue differences at otherresidue positions. In some embodiments, the number of differences can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26,30, 35, 40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO: 58 or 60), with the proviso that the engineered transaminasepolypeptide comprises polypeptide comprises an amino acid sequence thatincludes at least the features described for the specified residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence of SEQ ID NO:58 or 60.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X61 is an aromaticresidue, particularly Y; X62 is an aromatic or polar residue,particularly T, Y or F; X65 is an aliphatic residue, particularly A; X69is C or a non-polar, aliphatic or polar residue, particularly G, C, T,A, or S; X94 is an aliphatic residue, particularly I or L; X122 is aconstrained, non-polar or aliphatic residue, particularly M, I, L, V, orH; X136 is an aromatic residue, particularly Y or F; X137 is a polar oraliphatic residue, particularly T or I; X160 is an aliphatic residue,particularly L; X169 is an aliphatic residue, particularly L; X199 is analiphatic or aromatic residue, particularly W or I; X209 is an aliphaticresidue, particularly L; X215 is C; X223 is a constrained residue,particularly P; X269 is a constrained residue, particularly P; X282 is apolar residue, particularly S; and X284 is a non-polar residue,particularly G. In some embodiments, the transaminase polypeptides canhave additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11,1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40,1-45, 1-50, 1-55, or 1-60 residue differences at other residuepositions. In some embodiments, the number of differences can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35,40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO:62), with the proviso that the engineered transaminase polypeptidecomprises polypeptide comprises an amino acid sequence that includes atleast the features described for the specified residue positions. Insome embodiments, the engineered transaminase polypeptide can comprisean amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:62.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X61 is an aromaticresidue, particularly Y; X62 is an aromatic or polar residue,particularly T, Y or F; X65 is an aliphatic residue, particularly A; X69is C or a non-polar, aliphatic or polar residue, particularly G, C, T,A, or S; X94 is an aliphatic residue, particularly I or L; X122 is aconstrained, non-polar or aliphatic residue, particularly M, I, L, V, orH; X136 is an aromatic residue, particularly Y or F; X137 is a polar oraliphatic residue, particularly T or I; X169 is an aliphatic residue,particularly L; X199 is an aliphatic or aromatic residue, particularly Wor I; X209 is an aliphatic residue, particularly L; X215 is C; X223 is aconstrained residue, particularly P; X282 is a polar residue,particularly S; X284 is a non-polar residue, particularly G; and X306 isan aliphatic residue, particularly L. In some embodiments, thetransaminase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences at other residue positions. In some embodiments, the numberof differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences atthe other residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence based on SEQ IDNO:2 having the features described for preceding specified residuepositions (e.g., SEQ ID NO:64), with the proviso that the engineeredtransaminase polypeptide comprises polypeptide comprises an amino acidsequence that includes at least the features described for the specifiedresidue positions. In some embodiments, the engineered transaminasepolypeptide can comprise an amino acid sequence that is at least about80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% identical to a reference sequence of SEQ ID NO:64.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X61 is an aromaticresidue, particularly Y; X62 is an aromatic or polar residue,particularly T, Y or F; X65 is an aliphatic residue, particularly A; X69is C or a non-polar, aliphatic or polar residue, particularly G, C, T,A, or S; X94 is an aliphatic residue, particularly I or L; X102 is analiphatic or basic residue, particularly L or K; X122 is a constrained,non-polar or aliphatic residue, particularly M, I, L, V, or H; X136 isan aromatic residue, particularly Y or F; X150 is aromatic, constrainedor polar residue, particularly F, H, or S; X152 is C, non-polar,aliphatic, or polar residue, particularly G, I, L, S or C; X199 is analiphatic or aromatic residue, particularly W or I; X209 is an aliphaticresidue, particularly L; X215 is a C; X223 is a constrained residue,particularly P; X282 is a polar residue, particularly S; and X284 is anon-polar residue, particularly G. In some embodiments, the transaminasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residue differences at otherresidue positions. In some embodiments, the number of differences can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26,30, 35, 40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO:66), with the proviso that the engineered transaminase polypeptidecomprises polypeptide comprises an amino acid sequence that includes atleast the features described for the specified residue positions. Insome embodiments, the engineered transaminase polypeptide can comprisean amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:66.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X48 is a polar, acidic, aliphatic ornon-polar residue, particularly D, V, G, Q or A; X61 is an aromaticresidue, particularly Y; X62 is an aromatic or polar residue,particularly T, Y or F; X65 is an aliphatic residue, particularly A; X69is C or a non-polar, aliphatic or polar residue, particularly G, C, T,A, or S; X81 is a non-polar residue, particularly G; X94 is an aliphaticresidue, particularly I or L; X96 is an aliphatic residue, particularlyL; X102 is an aliphatic or basic residue, particularly L or K; X122 is aconstrained, non-polar or aliphatic residue, particularly M, I, L, V, orH; X136 is an aromatic residue, particularly Y or F; X163 is analiphatic or constrained residue, particularly H or V; X199 is analiphatic or aromatic residue, particularly W or I; X209 is an aliphaticresidue, particularly L; X211 is an aliphatic residue, particularly I;X215 is a C; X217 is a polar residue, particularly N; X223 is aconstrained residue, particularly P; X252 is an aromatic or aliphaticresidue, particularly F; X273 is an aromatic residue, particularly Y;X282 is a polar residue, particularly S; and X284 is a non-polarresidue, particularly G; and X321 is a constrained residue, particularlyP. In some embodiments, the transaminase polypeptides can haveadditionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45,1-50, 1-55, or 1-60 residue differences at other residue positions. Insome embodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50,55, or 60 residue differences at the other residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence based on SEQ ID NO:2 having the features describedfor preceding specified residue positions (e.g., SEQ ID NO:68), with theproviso that the engineered transaminase polypeptide comprisespolypeptide comprises an amino acid sequence that includes at least thefeatures described for the specified residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:68.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X48 is a polar, acidic, aliphatic ornon-polar residue, particularly A; X61 is an aromatic residue,particularly Y; X62 is an aromatic or polar residue, particularly T, Yor F; X65 is an aliphatic residue, particularly A; X69 is C or anon-polar, aliphatic or polar residue, particularly G, C, T, A, or S;X81 is a non-polar residue, particularly G; X94 is an aliphatic residue,particularly I or L; X122 is a constrained, non-polar or aliphaticresidue, particularly M, I, L, V, or H; X136 is an aromatic residue,particularly Y or F; X169 is an aliphatic residue, particularly L; X199is an aliphatic or aromatic residue, particularly W or I; X209 is analiphatic residue, particularly L; X215 is C; X217 is a polar residue,particularly N; X223 is a constrained residue, particularly P; X269 is aconstrained residue, particularly P; X282 is a polar residue,particularly S; X284 is a non-polar residue, particularly G; X297 is apolar residue, particularly S; and X321 is a constrained residue,particularly P. In some embodiments, the transaminase polypeptides canhave additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11,1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40,1-45, 1-50, 1-55, or 1-60 residue differences at other residuepositions. In some embodiments, the number of differences can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35,40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO:70), with the proviso that the engineered transaminase polypeptidecomprises an amino acid sequence that includes at least the featuresdescribed for the specified residue positions. In some embodiments, theengineered transaminase polypeptide can comprise an amino acid sequencethat is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% identical to a reference sequence of SEQ IDNO:70.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X61 is an aromatic residue, particularly Y;X62 is an aromatic or polar residue, particularly T, Y or F; X65 is analiphatic residue, particularly A; X69 is C or a non-polar, aliphatic orpolar residue, particularly G, C, T, A, or S; X94 is an aliphaticresidue, particularly I or L; X122 is a constrained, non-polar oraliphatic residue, particularly M, I, L, V, or H; X136 is an aromaticresidue, particularly Y or F; X199 is an aliphatic or aromatic residue,particularly W or I; X209 is an aliphatic residue, particularly L; X215is C; X223 is a constrained residue, particularly P; X282 is a polarresidue, particularly S; and X284 is a non-polar residue, particularlyG. In some embodiments, the transaminase polypeptides can haveadditionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45,1-50, 1-55, or 1-60 residue differences at other residue positions. Insome embodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50,55, or 60 residue differences at the other residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence based on SEQ ID NO:2 having the features describedfor preceding specified residue positions (e.g., SEQ ID NO:72), with theproviso that the engineered transaminase polypeptide comprises an aminoacid sequence that includes at least the features described for thespecified residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence of SEQ ID NO:72.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X61 is an aromatic residue, particularly Y;X62 is an aromatic or polar residue, particularly T, Y or F; X65 is analiphatic residue, particularly A; X69 is C or a non-polar, aliphatic orpolar residue, particularly G, C, T, A, or S; X81 is a non-polarresidue, particularly G; X94 is an aliphatic residue, particularly I orL; X96 is an aliphatic residue, particularly L; X122 is a constrained,non-polar or aliphatic residue, particularly M, I, L, V, or H; X136 isan aromatic residue, particularly Y or F; X178 is a polar residue,particularly S; X199 is an aliphatic or aromatic residue, particularly Wor I; X209 is an aliphatic residue, particularly L; X215 is C; X223 is aconstrained residue, particularly P; X269 is a constrained residue,particularly P; X282 is a polar residue, particularly S; X284 is anon-polar residue, particularly G; X297 is a polar residue, particularlyS; and X321 is a constrained residue, particularly P. In someembodiments, the transaminase polypeptides can have additionally 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or1-60 residue differences at other residue positions. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or60 residue differences at the other residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence based on SEQ ID NO:2 having the features describedfor preceding specified residue positions (e.g., SEQ ID NO:74), with theproviso that the engineered transaminase polypeptide comprises an aminoacid sequence that includes at least the features described for thespecified residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence of SEQ ID NO:74.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X60 is an aromatic residue, particularly F;X61 is an aromatic residue, particularly Y; X62 is an aromatic or polarresidue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X81 is a non-polar residue, particularlyG; X94 is an aliphatic residue, particularly I or L; X96 is an aliphaticresidue, particularly L; X122 is a constrained, non-polar or aliphaticresidue, particularly M, I, L, V, or H; X136 is an aromatic residue,particularly Y or F; X152 is C or a non-polar, aliphatic, or polarresidue, particularly G, I, L, S or C; X178 is a polar residue,particularly S; X199 is an aliphatic or aromatic residue, particularly Wor I; X209 is an aliphatic residue, particularly L; X215 is C; X217 is apolar residue, particularly N; X223 is a constrained residue,particularly P; X252 is an aromatic or aliphatic residue, particularlyF; X269 is a constrained residue, particularly P; X273 is an aromaticresidue, particularly Y; X282 is a polar residue, particularly S; andX284 is a non-polar residue, particularly G; X297 is a polar residue,particularly S; and X321 is a constrained residue, particularly P. Insome embodiments, the transaminase polypeptides can have additionally1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15,1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55,or 1-60 residue differences at other residue positions. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or60 residue differences at the other residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence based on SEQ ID NO:2 having the features describedfor preceding specified residue positions (e.g., SEQ ID NO:76), with theproviso that the engineered transaminase polypeptide comprises an aminoacid sequence that includes at least the features described for thespecified residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence of SEQ ID NO:76.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X60 is an aromatic residue, particularly F;X61 is an aromatic residue, particularly Y; X62 is an aromatic or polarresidue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X81 is a non-polar residue, particularlyG; X94 is an aliphatic residue, particularly I or L; X96 is an aliphaticresidue, particularly L; X122 is a constrained, non-polar or aliphaticresidue, particularly M, I, L, V, or H; X136 is an aromatic residue,particularly Y or F; X169 is an aliphatic residue, particularly L; X178is a polar residue, particularly S; X199 is an aliphatic or aromaticresidue, particularly W or I; X209 is an aliphatic residue, particularlyL; X215 is C; X217 is a polar residue, particularly N; X223 is aconstrained residue, particularly P; X269 is a constrained residue,particularly P; X282 is a polar residue, particularly S; and X284 is anon-polar residue, particularly G; X292 is a polar residue, particularlyT; X297 is a polar residue, particularly S; and X321 is a constrainedresidue, particularly P. In some embodiments, the transaminasepolypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8,1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26,1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residue differences at otherresidue positions. In some embodiments, the number of differences can be1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26,30, 35, 40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO:78), with the proviso that the engineered transaminase polypeptidecomprises an amino acid sequence that includes at least the featuresdescribed for the specified residue positions. In some embodiments, theengineered transaminase polypeptide can comprise an amino acid sequencethat is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% identical to a reference sequence of SEQ IDNO:78.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X60 is an aromatic residue, particularly F;X61 is an aromatic residue, particularly Y; X62 is an aromatic or polarresidue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X81 is a non-polar residue, particularlyG; X94 is an aliphatic residue, particularly I or L; X96 is an aliphaticresidue, particularly L; X122 is a constrained, non-polar or aliphaticresidue, particularly M, I, L, V, or H; X136 is an aromatic residue,particularly Y or F; X169 is an aliphatic residue, particularly L; X199is an aliphatic or aromatic residue, particularly W or I; X209 is analiphatic residue, particularly L; X215 is C; X217 is a polar residue,particularly N; X223 is a constrained residue, particularly P; X269 is aconstrained residue, particularly P; X273 is an aromatic residue,particularly Y; X282 is a polar residue, particularly S; and X284 is anon-polar residue, particularly G; X297 is a polar residue, particularlyS; and X321 is a constrained residue, particularly P. In someembodiments, the transaminase polypeptides can have additionally 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or1-60 residue differences at other residue positions. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or60 residue differences at the other residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence based on SEQ ID NO:2 having the features describedfor preceding specified residue positions (e.g., SEQ ID NO:80), with theproviso that the engineered transaminase polypeptide comprises an aminoacid sequence that includes at least the features described for thespecified residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence of SEQ ID NO:80.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X60 is an aromatic residue, particularly F;X61 is an aromatic residue, particularly Y; X62 is an aromatic or polarresidue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X81 is a non-polar residue, particularlyG; X94 is an aliphatic residue, particularly I or L; X96 is an aliphaticresidue, particularly L; X122 is a constrained, non-polar or aliphaticresidue, particularly M, I, L, V, or H; X136 is an aromatic residue,particularly Y or F; X169 is an aliphatic residue, particularly L; X178is a polar residue, particularly S; X199 is an aliphatic or aromaticresidue, particularly W or I; X209 is an aliphatic residue, particularlyL; X215 is C; X223 is a constrained residue, particularly P; X269 is aconstrained residue, particularly P; X273 is an aromatic residue,particularly Y; X282 is a polar residue, particularly S; and X284 is anon-polar residue, particularly G; X297 is a polar residue, particularlyS; and X321 is a constrained residue, particularly P. In someembodiments, the transaminase polypeptides can have additionally 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or1-60 residue differences at other residue positions. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or60 residue differences at the other residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence based on SEQ ID NO:2 having the features describedfor preceding specified residue positions (e.g., SEQ ID NO:82), with theproviso that the engineered transaminase polypeptide comprises an aminoacid sequence that includes at least the features described for thespecified residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence of SEQ ID NO:82

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X60 is an aromatic residue, particularly F;X61 is an aromatic residue, particularly Y; X62 is an aromatic or polarresidue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X81 is a non-polar residue, particularlyG; X94 is an aliphatic residue, particularly I or L; X96 is an aliphaticresidue, particularly L; X122 is a constrained, non-polar or aliphaticresidue, particularly M, I, L, V, or H; X124 is a polar or constrainedresidue, particularly T, H or N; X136 is an aromatic residue,particularly Y or F; X169 is an aliphatic residue, particularly L; X199is an aliphatic or aromatic residue, particularly W or I; X209 is analiphatic residue, particularly L; X215 is C; X217 is a polar residue,particularly N; X223 is a constrained residue, particularly P; X269 is aconstrained residue, particularly P; X273 is an aromatic residue,particularly Y; X282 is a polar residue, particularly S; and X284 is anon-polar residue, particularly G; X297 is a polar residue, particularlyS; and X321 is a constrained residue, particularly P. In someembodiments, the transaminase polypeptides can have additionally 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or1-60 residue differences at other residue positions. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or60 residue differences at the other residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence based on SEQ ID NO:2 having the features describedfor preceding specified residue positions (e.g., SEQ ID NO:84, 86, 88,96, 98, or 100), with the proviso that the engineered transaminasepolypeptide comprises an amino acid sequence that includes at least thefeatures described for the specified residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:84, 86, 88, 96, 98, or 100.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X60 is an aromatic residue, particularly F;X61 is an aromatic residue, particularly Y; X62 is an aromatic or polarresidue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X81 is a non-polar residue, particularlyG; X94 is an aliphatic residue, particularly I or L; X96 is an aliphaticresidue, particularly L; X122 is a constrained, non-polar or aliphaticresidue, particularly M, I, L, V, or H; X136 is an aromatic residue,particularly Y or F; X150 is aromatic, constrained or polar residue,particularly F, H, or S; X169 is an aliphatic residue, particularly L;X199 is an aliphatic or aromatic residue, particularly W or I; X209 isan aliphatic residue, particularly L; X215 is C; X217 is a polarresidue, particularly N; X223 is a constrained residue, particularly P;X269 is a constrained residue, particularly P; X273 is an aromaticresidue, particularly Y; X282 is a polar residue, particularly S; andX284 is a non-polar residue, particularly G; X297 is a polar residue,particularly S; and X321 is a constrained residue, particularly P. Insome embodiments, the transaminase polypeptides can have additionally1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15,1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55,or 1-60 residue differences at other residue positions. In someembodiments, the number of differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or60 residue differences at the other residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence based on SEQ ID NO:2 having the features describedfor preceding specified residue positions (e.g., SEQ ID NO:90), with theproviso that the engineered transaminase polypeptide comprises an aminoacid sequence that includes at least the features described for thespecified residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence of SEQ ID NO:90.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X60 is an aromatic residue, particularly F;X61 is an aromatic residue, particularly Y; X62 is an aromatic or polarresidue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X81 is a non-polar residue, particularlyG; X94 is an aliphatic residue, particularly I or L; X122 is aconstrained, non-polar or aliphatic residue, particularly M, I, L, V, orH; X124 is a polar or constrained residue, particularly T, H or N; X136is an aromatic residue, particularly Y or F; X150 is aromatic,constrained or polar residue, particularly F, H, or S; X152 is C or anon-polar, aliphatic, or polar residue, particularly G, I, L, S or C;X169 is an aliphatic residue, particularly L; X199 is an aliphatic oraromatic residue, particularly W or I; X209 is an aliphatic residue,particularly L; X215 is a C; X217 is a polar residue, particularly N;X223 is a constrained residue, particularly P; X269 is a constrainedresidue, particularly P; X273 is an aromatic residue, particularly Y;X282 is a polar residue, particularly S; and X284 is a non-polarresidue, particularly G; X297 is a polar residue, particularly S; andX321 is a constrained residue, particularly P. In some embodiments, thetransaminase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences at other residue positions. In some embodiments, the numberof differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences atthe other residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence based on SEQ IDNO:2 having the features described for preceding specified residuepositions (e.g., SEQ ID NO:92), with the proviso that the engineeredtransaminase polypeptide comprises an amino acid sequence that includesat least the features described for the specified residue positions. Insome embodiments, the engineered transaminase polypeptide can comprisean amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:92.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X60 is an aromatic residue, particularly F;X61 is an aromatic residue, particularly Y; X62 is an aromatic or polarresidue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X81 is a non-polar residue, particularlyG; X94 is an aliphatic residue, particularly I or L; X96 is an aliphaticresidue, particularly L; X122 is a constrained, non-polar or aliphaticresidue, particularly M, I, L, V, or H; X124 is a polar or constrainedresidue, particularly T, H or N; X136 is an aromatic residue,particularly Y or F; X150 is aromatic, constrained or polar residue,particularly F, H, or S; X152 is C or a non-polar, aliphatic, or polarresidue, particularly G, I, L, S or C; X169 is an aliphatic residue,particularly L; X199 is an aliphatic or aromatic residue, particularly Wor I; X209 is an aliphatic residue, particularly L; X215 is a C; X217 isa polar residue, particularly N; X223 is a constrained residue,particularly P; X269 is a constrained residue, particularly P; X273 isan aromatic residue, particularly Y; X282 is a polar residue,particularly S; and X284 is a non-polar residue, particularly G; X297 isa polar residue, particularly S; and X321 is a constrained residue,particularly P. In some embodiments, the transaminase polypeptides canhave additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11,1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40,1-45, 1-50, 1-55, or 1-60 residue differences at other residuepositions. In some embodiments, the number of differences can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35,40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO:94), with the proviso that the engineered transaminase polypeptidecomprises an amino acid sequence that includes at least the featuresdescribed for the specified residue positions. In some embodiments, theengineered transaminase polypeptide can comprise an amino acid sequencethat is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% identical to a reference sequence of SEQ IDNO:94.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X60 is an aromatic residue, particularly F;X61 is an aromatic residue, particularly Y; X62 is an aromatic or polarresidue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X81 is a non-polar residue, particularlyG; X94 is an aliphatic residue, particularly I or L; X96 is an aliphaticresidue, particularly L; X122 is a constrained, non-polar or aliphaticresidue, particularly M, I, L, V, or H; X124 is a polar or constrainedresidue, particularly T, H or N; X136 is an aromatic residue,particularly Y or F; X169 is an aliphatic residue, particularly L; X199is an aliphatic or aromatic residue, particularly W or I; X209 is analiphatic residue, particularly L; X215 is C; X217 is a polar residue,particularly N; X223 is a constrained residue, particularly P; X269 is aconstrained residue, particularly P; X273 is an aromatic residue,particularly Y; X282 is a polar residue, particularly S; and X284 is anon-polar residue, particularly G; X297 is a polar residue, particularlyS; X321 is a constrained residue, particularly P; and X329 is aconstrained or aromatic residue, particularly H. In some embodiments,the transaminase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5,1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20,1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences at other residue positions. In some embodiments, the numberof differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences atthe other residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence based on SEQ IDNO:2 having the features described for preceding specified residuepositions (e.g., SEQ ID NO:102), with the proviso that the engineeredtransaminase polypeptide comprises an amino acid sequence that includesat least the features described for the specified residue positions. Insome embodiments, the engineered transaminase polypeptide can comprisean amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:102.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X60 is an aromatic residue, particularly F;X61 is an aromatic residue, particularly Y; X62 is an aromatic or polarresidue, particularly T, Y or F; X65 is an aliphatic residue,particularly A; X69 is C or a non-polar, aliphatic or polar residue,particularly G, C, T, A, or S; X81 is a non-polar residue, particularlyG; X94 is an aliphatic residue, particularly I or L; X96 is an aliphaticresidue, particularly L; X122 is a constrained, non-polar or aliphaticresidue, particularly M, I, L, V, or H; X124 is a polar or constrainedresidue, particularly T, H or N; X136 is an aromatic residue,particularly Y or F; X150 is aromatic, constrained or polar residue,particularly S; X152 is cysteine (C), non-polar, aliphatic, or polarresidue, particularly G, I, L, S or C; X169 is an aliphatic residue,particularly L; X199 is an aliphatic or aromatic residue, particularly Wor I; X209 is an aliphatic residue, particularly L; X215 is C; X217 is apolar residue, particularly N; X223 is a constrained residue,particularly P; X269 is a constrained residue, particularly P; X273 isan aromatic residue, particularly Y; X282 is a polar residue,particularly S; X284 is a non-polar residue, particularly G; X297 is apolar residue, particularly S; and X321 is a constrained residue,particularly P. In some embodiments, the transaminase polypeptides canhave additionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11,1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40,1-45, 1-50, 1-55, or 1-60 residue differences at other residuepositions. In some embodiments, the number of differences can be 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24, 26, 30, 35,40, 45, 50, 55, or 60 residue differences at the other residuepositions. In some embodiments, the engineered transaminase polypeptidecan comprise an amino acid sequence that is at least about 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%identical to a reference sequence based on SEQ ID NO:2 having thefeatures described for preceding specified residue positions (e.g., SEQID NO:110), with the proviso that the engineered transaminasepolypeptide comprises an amino acid sequence that includes at least thefeatures described for the specified residue positions. In someembodiments, the engineered transaminase polypeptide can comprise anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:110.

In some embodiments, the engineered transaminase comprises an amino acidsequence that includes the following features: X8 is a constrainedresidue, particularly is P; X49 is a polar residue, particularly T; X60is an aromatic residue, particularly F; X61 is an aromatic residue,particularly Y; X62 is an aromatic or polar residue, particularly T, Yor F; X65 is an aliphatic residue, particularly A; X69 is C or anon-polar, aliphatic or polar residue, particularly G, C, T, A, or S;X81 is a non-polar residue, particularly G; X94 is an aliphatic residue,particularly I or L; X96 is an aliphatic residue, particularly L; X117is a non-polar residue, particularly G; X122 is a constrained, non-polaror aliphatic residue, particularly M, I, L, V, or H; X124 is a polar orconstrained residue, particularly T, H or N; X126 is a polar residue,particularly T; X136 is an aromatic residue, particularly Y or F; X150is aromatic, constrained or polar residue, particularly S; X152 iscysteine (C), non-polar, aliphatic, or polar residue, particularly G, I,L, S or C; X169 is an aliphatic residue, particularly L; X199 is analiphatic or aromatic residue, particularly W or I; X209 is an aliphaticresidue, particularly L; X215 is C; X217 is a polar residue,particularly N; X223 is a constrained residue, particularly P; X269 is aconstrained residue, particularly P; X273 is an aromatic residue,particularly Y; X282 is a polar residue, particularly S; X284 is anon-polar residue, particularly G; X297 is a polar residue, particularlyS; X302 is an aliphatic residue, particularly A; and X321 is aconstrained residue, particularly P. In some embodiments, thetransaminase polypeptides can have additionally 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22,1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences at other residue positions. In some embodiments, the numberof differences can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 residue differences atthe other residue positions. In some embodiments, the engineeredtransaminase polypeptide can comprise an amino acid sequence that is atleast about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% identical to a reference sequence based on SEQ IDNO:2 having the features described for preceding specified residuepositions (e.g., SEQ ID NO:166), with the proviso that the engineeredtransaminase polypeptide comprises an amino acid sequence that includesat least the features described for the specified residue positions. Insome embodiments, the engineered transaminase polypeptide can comprisean amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to areference sequence of SEQ ID NO:166.

Table 2 below provides exemplary engineered transaminase polypeptides,with each row listing two SEQ ID NOs, the odd number referring to thenucleotide sequence encoding the amino acid sequence provided by theeven number. The residue differences are based on comparison toreference sequence of SEQ ID NO:2, a transaminase derived fromArthrobacter sp KNK168 and differs from the naturally occurring enzymein having a substitution of isoleucine (I) at residue position X306 withvaline (V). In the Activity column, the levels of increasing activity(i.e., “+” “++” “+++” etc.) were defined as follows: “+” indicates atleast equal to but not greater than 2 times the activity of SEQ ID NO:4(assay conditions: 2 g/L ketoamide substrate, 0.5 M isopropylamine, 22°C., pH 7.5, 5% DMSO, 100 μM PLP); “++” indicates about 50-to-100 timesgreater than the activity of SEQ ID NO:4 (assay conditions: 2 g/Lketoamide substrate, 0.5 M isopropylamine, 22° C., pH 7.5, 5% MeOH, 100μM PLP); “+++” indicates about 1.1 to about 5 times greater than theactivity of SEQ ID NO:22 (assay conditions: 5-10 g/L ketoamidesubstrate, 0.5-1 M isopropylamine, 22-30° C., pH 7.5, 5% MeOH, 100 μMPLP); “++++” indicates about 1.1 to 5 times greater than the activity ofSEQ ID NO:48 (assay conditions: 10-40 g/L ketoamide substrate, 1 Misopropylamine, 30-45° C., pH 8.5, 10% MeOH, 100 μM PLP); “+++++”indicates about 1.1 to 5 times or greater than the activity of SEQ IDNO:58 (assay conditions: 40-100 g/L ketoamide substrate, 1 Misopropylamine, 45° C., pH 8.5, 10% MeOH-25% DMSO, 250 μM PLP); “++++++”indicates about 1.1 to 5 times or greater than the activity of SEQ IDNO:104 (assay conditions: 40-100 g/L ketoamide substrate, 1 Misopropylamine, 45° C., pH 8.5, 50% DMSO, 1000 μM PLP). Exemplary assayconditions for measuring activity using methanol and DMSO are describedin Examples 6-11.

TABLE 2 No. SEQ ID Residue NO Residue differences relative to SEQ ID NO:2 Differences Activity 1/2 — — −− 3/4 V69G; F122V; S223P; A284G 4 + 5/6V65A; V69G; F122I; S223P 4 + 7/8 F122L; S223P; A284G 3 +  9/10 F122I;S223P; A284G 3 + 11/12 F122L; S174A; S223P; A284G 4 + 13/14 Y26H; V65A;V69G; F122I; S223P; A284G 6 + 15/16 Y26H; H62T; V65A; V69G; F122I;T178S; V199W; S223P; 11 ++ F225Y; T282S; A284G 17/18 Y26H; H62F; V65A;V69G; F122V; G136Y; V199I; A209L; 12 ++ S223P; F225Y; T282S; A284G 19/20Y26H; H62T; V65A; V69G; F122V; G136Y; E137T; V199I; 12 ++ A209L; S223P;T282S; A284G 21/22 Y26H; H62T; V65A; V69G; F122I; G136Y; E137I; V199I;12 ++ A209L; S223P; T282S; A284G 23/24 Y26H; H62T; V65A; V69G; F122I;G136Y; E137T; V199I; 13 ++ A209L; S223P; F225Y; T282S; A284G 25/26 Y26H;V65A; V69G; F122V; G136Y; E137I; S174A; V199I; 12 ++ A209L; S223P;I230V; A284G 27/28 Y26H; H62T; V65A; V69G; F122H; G136Y; E137I; V199I;12 +++ A209L; S223P; T282S; A284G 29/30 Y26H; H62T; V65A; V69T; F122I;G136Y; E137I; V199I; 12 +++ A209L; S223P; T282S; A284G 31/32 Y26H; H62T;V65A; V69C; F122I; G136Y; E137I; V199I; 12 +++ A209L; S223P; T282S;A284G 33/34 Y26H; H62T; V65A; V69A; F122I; G136Y; E137I; V199I; 12 +++A209L; S223P; T282S; A284G 35/36 Y26H; L61Y; H62T; V65A; V69G; F122I;G136Y; E137I; 13 +++ V199I; A209L; S223P; T282S; A284G 37/38 Y26H; H62Y;V65A; V69G; F122I; G136Y; E137I; V199I; 12 +++ A209L; S223P; T282S;A284G 39/40 Y26H; H62T; V65A; V69G; F122I; G136F; E137I; V199I; 12 +++A209L; S223P; T282S; A284G 41/42 S4Y; Y26H; H62T; V65A; V69G; M94I;F122I; G136Y; 15 +++ E137T; V199I; A209L; G215C; S223P; T282S; A284G43/44 H62T; V65A; V69G; M94I; F122I; G136Y; E137I; V199I; 13 +++ A209L;G215C; S223P; T282S; A284G 45/46 H62T; V65A; V69G; M94I; F122I; G136Y;E137T; V199I; 13 +++ A209L; G215C; S223P; T282S; A284G 47/48 H62T; V65A;V69C; M94I; F122I; G136Y; E137T; V199I; 13 +++ A209L; G215C; S223P;T282S; A284G 49/50 S8P; H62T; V65A; V69C; M94I; F122I; G136Y; E137T; 14++++ V199I; A209L; G215C; S223P; T282S; A284G 51/52 L61Y; H62T; V65A;V69S; M94I; F122I; G136F; E137T; 15 ++++ V152I; V199I; A209L; G215C;S223P; T282S; A284G 53/54 L61Y; H62T; V65A; V69C; M94I; F122V; G136F;E137T; 14 ++++ V199I; A209L; G215C; S223P; T282S; A284G 55/56 L61Y;H62T; V65A; V69T; M94I; F122V; G136F; E137T; 14 ++++ V199I; A209L;G215C; S223P; T282S; A284G 57/58 L61Y; H62T; V65A; V69T; M94I; F122I;G136F; V199I; 13 ++++ A209L; G215C; S223P; T282S; A284G 59/60 L61Y;H62T; V65A; V69T; M94I; F122H; G136F; V199I; 13 ++++ A209L; G215C;S223P; T282S; A284G 61/62 L61Y; H62T; V65A; V69C; M94I; F122I; G136Y;E137T; 17 ++++ F160L; A169L; V199I; A209L; G215C; S223P; L269P; T282S;A284G 63/64 L61Y; H62T; V65A; V69C; M94L; F122I; G136Y; E137T; 16 ++++A169L; V199I; A209L; G215C; S223P; T282S; A284G; V306L 65/66 L61Y; H62T;V65A; V69C; M94I; Q102L; F122I; G136F; 16 ++++ Y150F; V152I; V199I;A209L; G215C; S223P; T282S; A284G 67/68 S8P; P48A; L61Y; H62T; V65A;V69T; D81G; M94I; I96L; 24 +++++ Q102K; F122I; G136F; I163V; V199I;A209L; L211I; G215C; G217N; S223P; L252F; L273Y; T282S; A284G; S321P69/70 S8P; P48A; L61Y; H62T; V65A; V69T; D81G; M94I; F122I; 21 +++++G136F; A169L; V199I; A209L; G215C; G217N; S223P; L269P; T282S; A284G;P297S; S321P 71/72 S8P; L61Y; H62T; V65A; V69T; M94I; F122I; G136F;V199I; 14 +++++ A209L; G215C; S223P; T282S; A284G 73/74 S8P; L61Y; H62T;V65A; V69T; D81G; M94L; I96L; F122I; 20 +++++ G136F; T178S; V199I;A209L; G215C; S223P; L269P; T282S; A284G; P297S; S321P 75/76 S8P; Y60F;L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 25 +++++ F122I; G136F; V152L;T178S; V199I; A209L; G215C; G217N; S223P; L252F; L269P; L273Y; T282S;A284G; P297S; S321P 77/78 S8P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94L;I96L; 24 +++++ F122I; G136F; A169L; T178S; V199I; A209L; G215C; G217N;S223P; L269P; T282S; A284G; S292T; P297S; S321P 79/80 S8P; Y60F; L61Y;H62T; V65A; V69T; D81G; M94I; I96L; 23 +++++ F122I; G136F; A169L; V199I;A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P81/82 S8P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94L; I96L; 23 +++++F122I; G136F; A169L; T178S; V199I; A209L; G215C; S223P; L269P; L273Y;T282S; A284G; P297S; S321P 83/84 S8P; Y60F; L61Y; H62T; V65A; V69T;D81G; M94I; I96L; 24 +++++ F122I; S124T; G136F; A169L; V199I; A209L;G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P 85/86 S8P;Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 24 +++++ F122I; S124H;G136F; A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y; T282S;A284G; P297S; S321P 87/88 S8P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I;I96L; 24 +++++ F122I; S124N; G136F; A169L; V199I; A209L; G215C; G217N;S223P; L269P; L273Y; T282S; A284G; P297S; S321P 89/90 S8P; Y60F; L61Y;H62T; V65A; V69T; D81G; M94I; I96L; 24 +++++ F122I; G136F; Y150H; A169L;V199I; A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S;S321P 91/92 S8P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 26+++++ F122M; S124H; G136F; Y150H; V152S; A169L; V199I; A209L; G215C;G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P 93/94 S8P; Y60F;L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 26 +++++ F122I; S124T; G136F;Y150S; V152C; A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y;T282S; A284G; P297S; S321P 95/96 S8P; Y60F; L61Y; H62T; V65A; V69T;D81G; M94I; I96L; 24 +++++ F122M; S124N; G136F; A169L; V199I; A209L;G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P 97/98 S8P;Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 24 +++++ F122I; S124H;G136F; A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y; T282S;A284G; P297S; S321P  99/100 S8P; Y60F; L61Y; H62T; V65A; V69T; D81G;M94I; I96L; 24 +++++ F122M; S124N; G136F; A169L; V199I; A209L; G215C;G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P 101/102 S8P;Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 25 +++++ F122M; S124N;G136F; A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y; T282S;A284G; P297S; S321P; Q329H 103/104 S8P; Y60F; L61Y; H62T; V65A; V69T;D81G; M94I; I96L; 26 +++++ F122M; S124T; G136F; Y150S; V152C; A169L;V199I; A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S;S321P 105/106 S8P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 29+++++ F122M; S124T; S126T; G136F; R138K; Y150S; V152G; Q155M; A169L;V199I; A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S;S321P 107/108 S8P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 28+++++ F122M; S124T; G136F; R138P; Q146R; Y150S; V152S; A169L; V199I;A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P109/110 S8P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 27 ++++++F122M; S124T; S126T; G136F; Y150S; V152C; A169L; V199I; A209L; G215C;G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P 111/112 S8P;Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 27 ++++++ F122M; S124T;G136F; Y150S; V152C; I163H; A169L; V199I; A209L; G215C; G217N; S223P;L269P; L273Y; T282S; A284G; P297S; S321P 113/114 S8P; Y60F; L61Y; H62T;V65A; V69T; D81G; M94I; I96L; 27 ++++++ F122M; S124T; G136F; Y148A;Y150S; V152C; A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y;T282S; A284G; P297S; S321P 115/116 S8P; Y60F; L61Y; H62T; V65A; V69T;D81G; M94I; I96L; 27 ++++++ F122M; S124T; G136F; Y150S; V152C; W156Q;A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G;P297S; S321P 117/118 S8P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I;I96L; 27 ++++++ F122M; S124T; G136F; Y150S; V152C; R164V; A169L; V199I;A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P119/120 S8P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 27 ++++++F122M; S124T; G136F; Y148F; Y150S; V152C; A169L; V199I; A209L; G215C;G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P 121/122 S8P;Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; I96L; 27 ++++++ E120Y; F122M;S124T; G136F; Y150S; V152C; A169L; V199I; A209L; G215C; G217N; S223P;L269P; L273Y; T282S; A284G; P297S; S321P 123/124 S8P; Y60F; L61Y; H62T;V65A; V69T; D81G; M94I; I96L; 27 ++++++ F122M; S124T; G136F; Y150S;V152C; Q155V; A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y;T282S; A284G; P297S; S321P 125/126 S8P; Y60F; L61Y; H62T; V65A; V69T;D81G; M94I; I96L; 27 ++++++ F122M; S124T; G136F; Y150S; V152C; R164P;A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G;P297S; S321P 127/128 S8P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I;I96L; 27 ++++++ F122M; S124T; G136F; Y150S; V152C; Q155T; A169L; V199I;A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P129/130 S8P; E50L; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; 27 ++++++I96L; F122M; S124T; G136F; Y150S; V152C; A169L; V199I; A209L; G215C;G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P 131/132 S8P;L18I; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; 27 ++++++ I96L; F122M;S124T; G136F; Y150S; V152C; A169L; V199I; A209L; G215C; G217N; S223P;L269P; L273Y; T282S; A284G; P297S; S321P 133/134 S8P; D25Q; Y60F; L61Y;H62T; V65A; V69T; D81G; M94I; 27 ++++++ I96L; F122M; S124T; G136F;Y150S; V152C; A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y;T282S; A284G; P297S; S321P 135/136 S8P; E42G; Y60F; L61Y; H62T; V65A;V69T; D81G; M94I; 27 ++++++ I96L; F122M; S124T; G136F; Y150S; V152C;A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G;P297S; S321P 137/138 S8P; P48D; Y60F; L61Y; H62T; V65A; V69T; D81G;M94I; 27 ++++++ I96L; F122M; S124T; G136F; Y150S; V152C; A169L; V199I;A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P139/140 S8P; P30Q; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; 27 ++++++I96L; F122M; S124T; G136F; Y150S; V152C; A169L; V199I; A209L; G215C;G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P 141/142 S8P;L28P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; 27 ++++++ I96L; F122M;S124T; G136F; Y150S; V152C; A169L; V199I; A209L; G215C; G217N; S223P;L269P; L273Y; T282S; A284G; P297S; S321P 143/144 S8P; I41H; Y60F; L61Y;H62T; V65A; V69T; D81G; M94I; 27 ++++++ I96L; F122M; S124T; G136F;Y150S; V152C; A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y;T282S; A284G; P297S; S321P 145/146 S8P; P30M; Y60F; L61Y; H62T; V65A;V69T; D81G; M94I; 27 ++++++ I96L; F122M; S124T; G136F; Y150S; V152C;A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G;P297S; S321P 147/148 S8P; S54H; Y60F; L61Y; H62T; V65A; V69T; D81G;M94I; 27 ++++++ I96L; F122M; S124T; G136F; Y150S; V152C; A169L; V199I;A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P149/150 S8P; L18C; I55V; Y60F; L61Y; H62T; V65A; V69T; D81G; 28 ++++++M94I; I96L; F122M; S124T; G136F; Y150S; V152C; A169L; V199I; A209L;G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P 151/152S8P; P48G; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; 27 ++++++ I96L;F122M; S124T; G136F; Y150S; V152C; A169L; V199I; A209L; G215C; G217N;S223P; L269P; L273Y; T282S; A284G; P297S; S321P 153/154 S8P; P48V; Y60F;L61Y; H62T; V65A; V69T; D81G; M94I; 27 ++++++ I96L; F122M; S124T; G136F;Y150S; V152C; A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y;T282S; A284G; P297S; S321P 155/156 S8P; I41S; Y60F; L61Y; H62T; V65A;V69T; D81G; M94I; 27 ++++++ I96L; F122M; S124T; G136F; Y150S; V152C;A169L; V199I; A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G;P297S; S321P 157/158 S8P; E27T; Y60F; L61Y; H62T; V65A; V69T; D81G;M94I; 27 ++++++ I96L; F122M; S124T; G136F; Y150S; V152C; A169L; V199I;A209L; G215C; G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P159/160 S8P; S54P; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; 27 ++++++I96L; F122M; S124T; G136F; Y150S; V152C; A169L; V199I; A209L; G215C;G217N; S223P; L269P; L273Y; T282S; A284G; P297S; S321P 161/162 S8P;P48Q; Y60F; L61Y; H62T; V65A; V69T; D81G; M94I; 27 ++++++ I96L; F122M;S124T; G136F; Y150S; V152C; A169L; V199I; A209L; G215C; G217N; S223P;L269P; L273Y; T282S; A284G; P297S; S321P 163/164 A5K; S8P; Y60F; L61Y;H62T; V65A; V69T; D81G; M94I; 28 ++++++ I96L; F122M; S124T; S126T;G136F; Y150S; V152C; A169L; V199I; A209L; G215C; G217N; S223P; L269P;L273Y; T282S; A284G; P297S; S321P 165/166 S8P; S49T; Y60F; L61Y; H62T;V65A; V69T; D81G; M94I; 30 ++++++ I96L; E117G; F122M; S124T; S126TG136F; Y150S; V152S; A169L; V199I; A209L; G215C; G217N; S223P; L269P;L273Y; T282S; A284G; P297S; V302A; S321P 167/168 S8P; S54P; Y60F; L61Y;H62T; V65A; V69T; D81G; M94I; 29 ++++++ I96L; F122M; S124T; S126T;G136F; Y150S; V152S; A169L; V199I; D204A; A209L; G215C; G217N; S223P;L269P; L273Y; T282S; A284G; P297S; S321P

As noted above, in some embodiments, the improved transaminasepolypeptide comprises an amino acid sequence that is at least about 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or more identical to a reference sequence of SEQ ID NO: 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168. Insome embodiments, the improved transaminase polypeptides can have 1-2,1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16,1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or1-60 residue differences as compared to the transaminase represented bySEQ ID NO:2. In some embodiments, the number of residue differences canbe 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24,26, 30, 35, 40, 45, 50, 55, or 60 differences as compared to SEQ IDNO:2.

In some embodiments, the improved transaminase polypeptide comprises anamino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to areference sequence based on SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122,124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,152, 154, 156, 158, 160, 162, 164, 166 or 168, with the proviso that theimproved transaminase amino acid sequence comprises any one of the setof residue differences contained in any one of the polypeptide sequenceslisted in Table 2 as compared to SEQ ID NO:2. In some embodiments, theimproved transaminase polypeptides can have additionally 1-2, 1-3, 1-4,1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20,1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, or 1-60 residuedifferences at other amino acid residue positions as compared to thereference sequence. In some embodiments, the number of differences canbe 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 18, 20, 22, 24,26, 30, 35, 40, 45, 50, 55, or 60 residue differences at other residuepositions. In some embodiments, the residue differences at other residuepositions comprise substitutions with conservative amino acid residues.

In some embodiments, the improved transaminase polypeptides capable ofconverting the ketoamide substrate,4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]1-(2,4,5-trifluorophenyl)butan-2-oneto product(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]1-(2,4,5-trifluorophenyl)butan-2-aminein the presence of an amino group donor to levels of product detectableby HPLC-UV at 210 nm comprises an amino acid sequence corresponding tothe sequence of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166 or 168.

In some embodiments, the engineered transaminase polypeptide is capableof converting the ketoamide substrate to product with 50 to 100 times orgreater activity than the polypeptide of SEQ ID NO:4. In someembodiments, the engineered transaminase polypeptide capable ofconverting the ketoamide substrate to product with 50 to 100 times orgreater activity than the polypeptide of SEQ ID NO:4 comprises an aminoacid sequence corresponding to SEQ ID NO: 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166 or 168.

In some embodiments, the engineered transaminase polypeptide is capableof converting the ketoamide substrate to product with about 1.1 to 5times or greater activity than the polypeptide of SEQ ID NO:22. In someembodiments, the engineered transaminase polypeptide capable ofconverting the ketoamide substrate to product with about 1.1 to 5 timesor greater activity than the polypeptide of SEQ ID NO:22 comprises anamino acid sequence corresponding to the sequence of SEQ ID NO: 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166 or 168.

In some embodiments, the engineered transaminase polypeptide is capableof converting the ketoamide substrate to product with about1.1-to-5-times or greater activity than the polypeptide of SEQ ID NO:48.In some embodiments, the engineered transaminase polypeptide capable ofconverting the ketoamide substrate to product with about 1.1-to-5-timesor greater activity than the polypeptide of SEQ ID NO:48 comprises asequence corresponding to the sequence of SEQ ID NO: 50, 52, 54, 56, 58,60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94,96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166 or 168.

In some embodiments, the engineered transaminase polypeptide is capableof converting the ketoamide substrate to product with about 1.1 to 5times or greater activity than the polypeptide of SEQ ID NO:58. In someembodiments, the engineered transaminase polypeptide capable ofconverting the ketoamide substrate to product with about 1.1 to 5 timesor greater activity than the polypeptide of SEQ ID NO:58 comprises anamino acid sequence corresponding to the sequence of SEQ ID NO: 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,162, 164, 166 or 168.

As noted above, in some embodiments, the improved transaminasepolypeptide is also capable of converting ketoamide substrate4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]1-(2,4,5-trifluorophenyl)butan-2-oneto product(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-aminewith at least 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% enantiomeric excess. Exemplary transaminase polypeptideswith the specified levels of enantioselectivity can comprise an aminoacid sequence corresponding to SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120,122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,150, 152, 154, 156, 158, 160, 162, 164, 166 or 168.

In some embodiments, the improved engineered transaminase polypeptidescan comprise deletions of the engineered transaminase polypeptidesdescribed herein. Thus, for each and every embodiment of thetransaminase polypeptides of the disclosure, the deletions can compriseone or more amino acids, 2 or more amino acids, 3 or more amino acids, 4or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 ormore amino acids, 10 or more amino acids, 15 or more amino acids, or 20or more amino acids, up to 10% of the total number of amino acids, up to10% of the total number of amino acids, up to 20% of the total number ofamino acids, or up to 30% of the total number of amino acids of thetransaminase polypeptides, as long as the functional activity of thetransaminase activity is maintained. In some embodiments, the deletionscan comprise, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12,1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45,1-50, 1-55, or 1-60 amino acid residues. In some embodiments, the numberof deletions can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16,18, 20, 22, 24, 26, 30, 35, 40, 45, 50, 55, or 60 amino acids. In someembodiments, the deletions can comprise deletions of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 22, 24, 26, 28, or 30 aminoacid residues.

As described herein, the transaminase polypeptides of the disclosure canbe in the form of fusion polypeptides in which the transaminasepolypeptides are fused to other polypeptides, such as, by way of exampleand not limitation, antibody tags (e.g., myc epitope), purificationssequences (e.g., His tags for binding to metals), and cell localizationsignals (e.g., secretion signals). Thus, the transaminase polypeptidescan be used with or without fusions to other polypeptides.

The polypeptides described herein are not restricted to the geneticallyencoded amino acids. In addition to the genetically encoded amino acids,the polypeptides described herein may be comprised, either in whole orin part, of naturally-occurring and/or synthetic non-encoded aminoacids. Certain commonly encountered non-encoded amino acids of which thepolypeptides described herein may be comprised include, but are notlimited to: the D-stereoisomers of the genetically-encoded amino acids;2,3-diaminopropionic acid (Dpr); α-aminoisobutyric acid (Aib);ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycineor sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit);t-butylalanine (Bua); t-butylglycine (Bug); N-methylisoleucine (MeIle);phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle);naphthylalanine (Nal); 2-chlorophenylalanine (Ocf);3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf);2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff);4-fluorophenylalanine (Pff); 2-bromophenylalanine (Obf);3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf);2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf);4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf);3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf);2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf);4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf);3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine(Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif);4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef);3,4-dichlorophenylalanine (Mpcf); 2,4-difluorophenylalanine (Opff);3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla);pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine(1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla);benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla);homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp);pentafluorophenylalanine (5ff); styrylkalanine (sAla); authrylalanine(aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenypentanoic acid (Afp);penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid(Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso);N(w)-nitroarginine (nArg); homolysine (hLys);phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer);phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutamic acid(hGlu); 1-aminocyclopent-(2 or 3)-ene-4 carboxylic acid; pipecolic acid(PA), azetidine-3-carboxylic acid (ACA);1-aminocyclopentane-3-carboxylic acid; allylglycine (aOly);propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal);homoleucine (hLeu), homovaline (hVal); homoisoleucine (hIle);homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid(Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal);homocysteine (hCys); homoserine (hSer); hydroxyproline (Hyp) andhomoproline (hPro). Additional non-encoded amino acids of which thepolypeptides described herein may be comprised will be apparent to thoseof skill in the art (see, e.g., the various amino acids provided inFasman, 1989, CRC Practical Handbook of Biochemistry and MolecularBiology, CRC Press, Boca Raton, Fla., at pp. 3-70 and the referencescited therein, all of which are incorporated by reference). These aminoacids may be in either the L- or D-configuration.

Those of skill in the art will recognize that amino acids or residuesbearing side chain protecting groups may also comprise the polypeptidesdescribed herein. Non-limiting examples of such protected amino acids,which in this case belong to the aromatic category, include (protectinggroups listed in parentheses), but are not limited to: Arg(tos),Cys(methylbenzyl), Cys (nitropyridinesulfenyl), Glu(δ-benzylester),Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos),Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr (O-benzyl) and Tyr(O-benzyl).

Non-encoding amino acids that are conformationally constrained of whichthe polypeptides described herein may be composed include, but are notlimited to, N-methyl amino acids (L-configuration); 1-aminocyclopent-(2or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylicacid; homoproline (hPro); and 1-aminocyclopentane-3-carboxylic acid.

As described above the various modifications introduced into thenaturally occurring polypeptide to generate an engineered transaminaseenzyme can be targeted to a specific property of the enzyme.

In another aspect, the present disclosure provides polynucleotidesencoding the improved transaminase polypeptides. The polynucleotides maybe operatively linked to one or more heterologous regulatory sequencesthat control gene expression to create a recombinant polynucleotidecapable of expressing the transaminase polypeptide. Expressionconstructs containing a heterologous polynucleotide encoding theengineered transaminase can be introduced into appropriate host cells toexpress the corresponding transaminase polypeptide.

Because of the knowledge of the codons corresponding to the variousamino acids, availability of a protein sequence provides a descriptionof all the polynucleotides capable of encoding the subject. Thedegeneracy of the genetic code, where the same amino acids are encodedby alternative or synonymous codons allows an extremely large number ofnucleic acids to be made, all of which encode the improved transaminasepolypeptides disclosed herein. Thus, having identified a particularamino acid sequence, those skilled in the art could make any number ofdifferent nucleic acids by simply modifying the sequence of one or morecodons in a way which does not change the amino acid sequence of theprotein. In this regard, the present disclosure specificallycontemplates each and every possible variation of polynucleotides thatcould be made by selecting combinations based on the possible codonchoices, and all such variations are to be considered specificallydisclosed for any polypeptide disclosed herein, including the amino acidsequences presented in Table 2.

In some embodiments, the polynucleotides can be selected and/orengineered to comprise codons that are preferably selected to fit thehost cell in which the protein is being produced. For example, preferredcodons used in bacteria are used to express the gene in bacteria;preferred codons used in yeast are used for expression in yeast; andpreferred codons used in mammals are used for expression in mammaliancells. Since not all codons need to be replaced to optimize the codonusage of the transaminases (e.g., because the natural sequence can havepreferred codons and because use of preferred codons may not be requiredfor all amino acid residues), codon optimized polynucleotides encodingthe transaminase polypeptides may contain preferred codons at about 40%,50%, 60%, 70%, 80%, or greater than 90% of codon positions of the fulllength coding region.

In some embodiments, the polynucleotide encodes a transaminasepolypeptide comprising an amino acid sequence that is at least 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% ormore identical to the reference sequence of SEQ ID NO:4, wherein thepolypeptide is capable of converting the ketoamide substrate4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-oneto product(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-aminein the presence of an amino group donor with an activity that isimproved as compared to the activity of the transaminase of SEQ ID NO:2derived from Arthrobacter sp KNK168.

In some embodiments, the polynucleotide encodes a transaminasepolypeptide comprising an amino acid sequence that has at least about80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more sequence identity to the polypeptide comprising anamino acid sequence corresponding to SEQ ID NO: 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, or 102, wherein the polypeptide has one or moreimproved properties in converting the ketoamide substrate4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-oneto product(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]1-(2,4,5-trifluorophenyl)butan-2-aminein presence of an amino group donor. In some embodiments, the encodedtransaminase polypeptide has an activity that is equal to or greaterthan the activity of the polypeptide of SEQ ID NO:4.

In some embodiments, the polynucleotide encodes a transaminasepolypeptide comprising an amino acid sequence that is at least about80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a reference sequence of SEQ ID NO: 4, 6, 8, 10,12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82,84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114,116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142,144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168.

In some embodiments, the polynucleotide encodes a transaminasepolypeptide comprising an amino acid sequence that is at least about80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identical to a reference sequence based on SEQ ID NO: 4, 6,8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78,80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110,112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138,140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or168, with the proviso that the improved transaminase amino acid sequencecomprises any one of the set of residue differences contained in any oneof the polypeptide sequences listed in Table 2 as compared to SEQ IDNO:2.

In some embodiments, the polynucleotides encoding the improvedtransaminase polypeptides are selected from SEQ ID NO: 3, 5, 7, 9, 11,13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83,85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143,145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, or 167.

In some embodiments, the polynucleotides are capable of hybridizingunder highly stringent conditions to a polynucleotide comprising SEQ IDNO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73,75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107,109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135,137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,165, or 167, or a complement thereof, where the highly stringentlyhybridizing polynucleotides encode a transaminase polypeptide capable ofconverting4-oxo-443-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-oneto product (2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-aminein presence of an amino group donor with an activity that is equal to orgreater than the polypeptide of SEQ ID NO:4.

In some embodiments, the polynucleotides encode the polypeptidesdescribed herein but have about 80% or more sequence identity, about80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% or more sequence identity at the nucleotide level to areference polynucleotide encoding the engineered transaminase describedherein. In some embodiments, the reference polynucleotide is selectedfrom SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67,69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, or 167.

An isolated polynucleotide encoding an improved transaminase polypeptidemay be manipulated in a variety of ways to provide for expression of thepolypeptide. In some embodiments, the polynucleotides encoding theengineered transaminase polypeptides can be provided as expressionvectors where one or more control sequences is present to regulate theexpression of the polynucleotides. Manipulation of the isolatedpolynucleotide prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotides and nucleic acid sequences utilizingrecombinant DNA methods are well known in the art. Guidance is providedin Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rdEd., Cold Spring Harbor Laboratory Press; and Current Protocols inMolecular Biology, Ausubel. F. ed., Greene Pub. Associates, 1998,updates to 2006.

In some embodiments, the control sequences include among others,promoters, leader sequence, polyadenylation sequence, propeptidesequence, signal peptide sequence, and transcription terminator. Forbacterial host cells, suitable promoters for directing transcription ofthe nucleic acid constructs of the present disclosure, include thepromoters obtained from the E. coli lac operon, E. coli trp operon,bacteriophage λ, Streptomyces coelicolor agarase gene (dagA), Bacillussubtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylasegene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proc. Natl Acad. Sci. USA 75: 3727-3731), as well as the tac promoter(DeBoer et al., 1983, Proc. Natl Acad. Sci. USA 80: 21-25).

For filamentous fungal host cells, suitable promoters for directing thetranscription of the nucleic acid constructs of the present disclosureinclude promoters obtained from the genes for Aspergillus oryzae TAKAamylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, andFusarium oxysporum trypsin-like protease (see e.g., WO 96/00787, whichis hereby incorporated by reference herein), as well as the NA2-tpipromoter (a hybrid of the promoters from the genes for Aspergillus nigerneutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase), and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters can be from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), andSaccharomyces cerevisiae 3-phosphoglycerate kinase. Other usefulpromoters for yeast host cells are described by Romanos et al., 1992,Yeast 8:423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

For example, exemplary transcription terminators for filamentous fungalhost cells can be obtained from the genes for Aspergillus oryzae TAKAamylase, Aspergillus niger glucoamylase, Aspergillus nidulansanthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusariumoxysporum trypsin-like protease.

Exemplary terminators for yeast host cells can be obtained from thegenes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA that is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleic acid sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used. Exemplaryleaders for filamentous fungal host cells are obtained from the genesfor Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells are obtainedfrom the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleic acid sequence andwhich, when transcribed, is recognized by the host cell as a signal toadd polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention. Exemplary polyadenylation sequences forfilamentous fungal host cells can be from the genes for Aspergillusoryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillusnidulans anthranilate synthase, Fusarium oxysporum trypsin-likeprotease, and Aspergillus niger alpha-glucosidase. Usefulpolyadenylation sequences for yeast host cells are described by Guo andSherman, 1995, Mol Cell Bio 15:5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion that encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region thatis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NC1B11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiol Rev 57: 109-137.

Effective signal peptide coding regions for filamentous fungal hostcells can be the signal peptide coding regions obtained from the genesfor Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells can be from the genes forSaccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalactase (see e.g., WO 95/33836, which is hereby incorporated byreference herein).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences, which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. In prokaryotic host cells, suitable regulatory sequencesinclude the lac, tac, and trp operator systems. In yeast host cells,suitable regulatory systems include, as examples, the ADH2 system orGAL1 system. In filamentous fungi, suitable regulatory sequences includethe TAKA alpha-amylase promoter, Aspergillus niger glucoamylasepromoter, and Aspergillus oryzae glucoamylase promoter.

Other examples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene, which is amplified in the presence of methotrexate, andthe metallothionein genes, which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the transaminasepolypeptide of the present invention would be operably linked with theregulatory sequence.

Thus, in another embodiment, the present disclosure is also directed toa recombinant expression vector comprising a polynucleotide encoding anengineered transaminase polypeptide or a variant thereof, and one ormore expression regulating regions such as a promoter and a terminator,a replication origin, etc., depending on the type of hosts into whichthey are to be introduced. The various nucleic acid and controlsequences described above may be joined together to produce arecombinant expression vector which may include one or more convenientrestriction sites to allow for insertion or substitution of the nucleicacid sequence encoding the polypeptide at such sites. Alternatively, thenucleic acid sequence of the present disclosure may be expressed byinserting the nucleic acid sequence or a nucleic acid constructcomprising the sequence into an appropriate vector for expression. Increating the expression vector, the coding sequence is located in thevector so that the coding sequence is operably linked with theappropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the polynucleotidesequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.

The expression vector may be an autonomously replicating vector, i.e., avector that exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The expression vector of the present invention preferably contains oneor more selectable markers, which permit easy selection of transformedcells. A selectable marker is a gene the product of which provides forbiocide or viral resistance, resistance to heavy metals, prototrophy toauxotrophs, and the like. Examples of bacterial selectable markers arethe dal genes from Bacillus subtilis or Bacillus licheniformis, ormarkers, which confer antibiotic resistance such as ampicillin,kanamycin, chloramphenicol, or tetracycline resistance. Suitable markersfor yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

Selectable markers for use in a filamentous fungal host cell include,but are not limited to, amdS (acetamidase), argB (ornithinecarbamoyltransferase), bar (phosphinothricin acetyltransferase), hph(hygromycin phosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Embodiments for use in an Aspergillus cell include the amdS and pyrGgenes of Aspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The expression vectors for expressing the transaminases can contain anelement(s) that permits integration of the vector into the host cell'sgenome or autonomous replication of the vector in the cell independentof the genome. For integration into the host cell genome, the vector mayrely on the nucleic acid sequence encoding the polypeptide or any otherelement of the vector for integration of the vector into the genome byhomologous or nonhomologous recombination.

Alternatively, the expression vector may contain additional nucleic acidsequences for directing integration by homologous recombination into thegenome of the host cell. The additional nucleic acid sequences enablethe vector to be integrated into the host cell genome at a preciselocation(s) in the chromosome(s). To increase the likelihood ofintegration at a precise location, the integrational elements shouldpreferably contain a sufficient number of nucleic acids, such as 100 to10,000 base pairs, preferably 400 to 10,000 base pairs, and mostpreferably 800 to 10,000 base pairs, which are highly homologous withthe corresponding target sequence to enhance the probability ofhomologous recombination. The integrational elements may be any sequencethat is homologous with the target sequence in the genome of the hostcell. Furthermore, the integrational elements may be non-encoding orencoding nucleic acid sequences. On the other hand, the vector may beintegrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are P15Aori, or the origins of replication of plasmids pBR322, pUC19, pACYC177(which plasmid has the P15A ori), or pACYC184 permitting replication inE. coli, and pUB110, pE194, pTA1060, or pAMβ1 permitting replication inBacillus. Examples of origins of replication for use in a yeast hostcell are the 2 micron origin of replication, ARS1, ARS4, the combinationof ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin ofreplication may be one having a mutation which makes its functioningtemperature-sensitive in the host cell (see, e.g., Ehrlich, 1978, ProcNatl Acad Sci. USA 75:1433).

More than one copy of a nucleic acid sequence of the present inventionmay be inserted into the host cell to increase production of the geneproduct. An increase in the copy number of the nucleic acid sequence canbe obtained by integrating at least one additional copy of the sequenceinto the host cell genome or by including an amplifiable selectablemarker gene with the nucleic acid sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleic acid sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent.

Many of the expression vectors for use in the present invention arecommercially available. Suitable commercial expression vectors includep3×FLAGTMTM expression vectors from Sigma-Aldrich Chemicals, St. LouisMo., which includes a CMV promoter and hGH polyadenylation site forexpression in mammalian host cells and a pBR322 origin of replicationand ampicillin resistance markers for amplification in E. coli. Othersuitable expression vectors are pBluescriptII SK(-) and pBK-CMV, whichare commercially available from Stratagene, LaJolla Calif., and plasmidswhich are derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pREP4, pCEP4(Invitrogen) or pPoly (Lathe et al., 1987, Gene 57:193-201).

In another aspect, the present disclosure provides a host cellcomprising a polynucleotide encoding an improved transaminasepolypeptide of the present disclosure, the polynucleotide beingoperatively linked to one or more control sequences for expression ofthe transaminase enzyme in the host cell. Host cells for use inexpressing the transaminase polypeptides encoded by the expressionvectors of the present invention are well known in the art and includebut are not limited to, bacterial cells, such as E. coli, Lactobacillus,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCCAccession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowesmelanoma cells; and plant cells. Appropriate culture mediums and growthconditions for the above-described host cells are well known in the art.

Polynucleotides for expression of the transaminase may be introducedinto cells by various methods known in the art. Techniques include amongothers, electroporation, biolistic particle bombardment, liposomemediated transfection, calcium chloride transfection, and protoplastfusion. Various methods for introducing polynucleotides into cells willbe apparent to the skilled artisan.

An exemplary host cell is Escherichia coli W3110. The expression vectorwas created by operatively linking a polynucleotide encoding an improvedtransaminase into the plasmid pCK110900 operatively linked to the lacpromoter under control of the lad repressor. The expression vector alsocontained the P15a origin of replication and the chloramphenicolresistance gene. Cells containing the subject polynucleotide inEscherichia coli W3110 were isolated by subjecting the cells tochloramphenicol selection.

The improved transaminases and polynucleotides encoding suchpolypeptides can be prepared using methods commonly used by thoseskilled in the art. As noted above, the naturally-occurring amino acidsequence and corresponding polynucleotide encoding the wild typetransaminase enzyme of Arthrobacter sp KNK168, from which the parentsequence SEQ ID NO:2 was derived, is available in U.S. Pat. No.7,169,592, which is hereby incorporated by reference herein. In someembodiments, the parent polynucleotide sequence is codon optimized toenhance expression of the transaminase in a specified host cell. Thepolynucleotide sequence designated SEQ ID NO: 1 was the parent sequenceutilized as the starting point for most experiments and libraryconstruction of engineered transaminases.

The engineered transaminases can be obtained by subjecting thepolynucleotide encoding the naturally occurring transaminase tomutagenesis and/or directed evolution methods. An exemplary directedevolution technique is mutagenesis and/or DNA shuffling as described inStemmer, 1994, Proc Natl Acad Sci USA 91:10747-10751; WO 95/22625; WO97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767 and U.S.Pat. No. 6,537,746 (each of which is hereby incorporated by referenceherein).

Other directed evolution procedures that can be used include, amongothers, staggered extension process (StEP), in vitro recombination (Zhaoet al., 1998, Nat. Biotechnol. 16:258-261), mutagenic PCR (Caldwell etal., 1994, PCR Methods Appl. 3:S136-S140), and cassette mutagenesis(Black et al., 1996, Proc Natl Acad Sci USA 93:3525-3529). Mutagenesisand directed evolution techniques useful for the purposes herein arealso described in the following references: Ling, et al., 1997,“Approaches to DNA mutagenesis: an overview,” Anal. Biochem.254(2):157-78; Dale et al., 1996, “Oligonucleotide-directed randommutagenesis using the phosphorothioate method,” Methods Mol. Biol.57:369-74; Smith, 1985, “In vitro mutagenesis,” Ann. Rev. Genet.19:423-462; Botstein et al., 1985, “Strategies and applications of invitro mutagenesis,” Science 229:1193-1201; Carter, 1986, “Site-directedmutagenesis,” Biochem. J. 237:1-7; Kramer et al., 1984, “Point MismatchRepair,” Cell 38:879-887; Wells et al., 1985, “Cassette mutagenesis: anefficient method for generation of multiple mutations at defined sites,”Gene 34:315-323; Minshull et al., 1999, “Protein evolution by molecularbreeding,” Curr Opin Chem Biol 3:284-290; Christians et al., 1999,“Directed evolution of thymidine kinase for AZT phosphorylation usingDNA family shuffling,” Nature Biotech 17:259-264; Crameri et al., 1998,“DNA shuffling of a family of genes from diverse species acceleratesdirected evolution,” Nature 391:288-291; Crameri et al., 1997,“Molecular evolution of an arsenate detoxification pathway by DNAshuffling,” Nature Biotech 15:436-438; Zhang et al., 1997, “Directedevolution of an effective fructosidase from a galactosidase by DNAshuffling and screening,” Proc Natl Acad Sci USA 94:45-4-4509; Crameriet al., 1996, “Improved green fluorescent protein by molecular evolutionusing DNA shuffling,' Nature Biotech 14:315-319; and Stemmer, 1994,“Rapid evolution of a protein in vitro by DNA shuffling,” Nature370:389-391. All publications are incorporated herein by reference.

In some embodiments, the clones obtained following mutagenesis treatmentare screened for transaminases having a desired improved enzymeproperty. Measuring transaminase enzyme activity from the expressionlibraries can be performed using the standard techniques, such asseparation of the product (e.g., by HPLC) and detection of the productby measuring UV absorbance of the separated substrate and productsand/or by detection using tandem mass spectroscopy (e.g., MS/MS).Exemplary assays are described in Example 4 below. The rate of increasein desired product per unit time indicates the relative (enzymatic)activity of the transaminase polypeptide in a fixed amount of the lysate(or a lyophilized powder made therefrom). Where the improved enzymeproperty desired is thermal stability, enzyme activity may be measuredafter subjecting the enzyme preparations to a defined temperature andmeasuring the amount of enzyme activity remaining after heat treatments.Clones containing a polynucleotide encoding the desired transaminasesare then isolated, sequenced to identify the nucleotide sequence changes(if any), and used to express the enzyme in a host cell.

Where the sequence of the engineered polypeptide is known, thepolynucleotides encoding the enzyme can be prepared by standardsolid-phase methods, according to known synthetic methods. In someembodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical litigationmethods, or polymerase mediated methods) to form any desired continuoussequence. For example, polynucleotides and oligonucleotides of theinvention can be prepared by chemical synthesis using, e.g., theclassical phosphoramidite method described by Beaucage et al., 1981, TetLett 22:1859-69, or the method described by Matthes et al., 1984, EMBOJ. 3:801-05, e.g., as it is typically practiced in automated syntheticmethods. According to the phosphoramidite method, oligonucleotides aresynthesized, e.g., in an automatic DNA synthesizer, purified, annealed,ligated and cloned in appropriate vectors. In addition, essentially anynucleic acid can be obtained from any of a variety of commercialsources, The Great American Gene Company, Ramona, Calif., ExpressGenInc. Chicago, Ill., Operon Technologies Inc., Alameda, Calif., and manyothers.

The engineered transaminase enzymes expressed in a host cell can berecovered from the cells and or the culture medium using any one or moreof the well known techniques for protein purification, including, amongothers, lysozyme treatment, sonication, filtration, salting-out,ultra-centrifugation, and chromatography. Suitable solutions for lysingand the high efficiency extraction of proteins from bacteria, such as E.coli., are commercially available under the trade name CelLytic BTM fromSigma-Aldrich of St. Louis Mo.

Chromatographic techniques for isolation of the transaminase polypeptideinclude, among others, reverse phase chromatography high performanceliquid chromatography, ion exchange chromatography, gel electrophoresis,and affinity chromatography. Conditions for purifying a particularenzyme will depend, in part, on factors such as net charge,hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc.,and will be apparent to those having skill in the art. In someembodiments, the engineered transaminases can be expressed as fusionproteins with purification tags, such as His-tags having affinity formetals, or antibody tags for binding to antibodies, e.g., myc epitopetag.

In some embodiments, affinity techniques may be used to isolate theimproved transaminase enzymes. For affinity chromatography purification,any antibody which specifically binds the transaminase polypeptide maybe used. For the production of antibodies, various host animals,including but not limited to rabbits, mice, rats, etc., may be immunizedby injection with an engineered polypeptide. The polypeptide may beattached to a suitable carrier, such as BSA, by means of a side chainfunctional group or linkers attached to a side chain functional group.Various adjuvants may be used to increase the immunological response,depending on the host species, including but not limited to Freund's(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(bacilli Calmette Guerin) and Corynebacterium parvum.

In a further aspect, the improved transaminase polypeptides describedherein can be used in a process for transamination of certain aminogroup acceptors (e.g., a ketone acceptor) in presence of an amino groupdonor. For the description of the compounds herein, the followingmeanings shall apply.

“Alkyl” is intended to include alkyl groups of the designated length ineither a straight or branched configuration. Exemplary of such alkylgroups are methyl, ethyl, propyl, isopropyl, butyl, sec-butyl,tert-butyl, pentyl, isopentyl, hexyl, isohexyl, and the like. The alkylgroups are unsubstituted or substituted with one to three groupsindependently selected from the group consisting of halogen, hydroxy,carboxy, aminocarbonyl, amino, C₁₋₄ alkoxy, and C₁₋₄ alkylthio.

“Cycloalkyl” is intended to mean cyclic rings of alkanes of five totwelve total carbon atoms, or any number within this range (i.e.,cyclopentyl, cyclohexyl, cycloheptyl, etc).

“Halogen” is intended to include the halogen atoms fluorine, chlorine,bromine, and iodine.

“Aryl” is intended to mean an aromatic group, including phenyl andnaphthyl. “Aryl” is unsubstituted or substituted with one to fivesubstituents independently selected from fluoro, hydroxy,trifluoromethyl, amino, C₁₋₄ alkyl, and C₁₋₄ alkoxy.

“Heteroaryl” means an 5- or 6-membered aromatic heterocycle thatcontains at least one ring heteroatom selected from O, S and N.Heteroaryls also include heteroaryls fused to other kinds of rings, suchas aryls, cycloalkyls and heterocycles that are not aromatic. Examplesof heteroaryl groups include, but are not limited to, pyrrolyl,isoxazolyl, isothiazolyl, pyrazolyl, pyridinyl, oxazolyl,1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, thiadiazolyl, thiazolyl,imidazolyl, triazolyl, tetrazolyl, furyl, triazinyl, thienyl,pyrimidinyl, pyrazinyl, benzisoxazolyl, benzoxazolyl, benzothiazolyl,benzothiadiazolyl, dihydrobenzofuranyl, indolinyl, pyridazinyl,indazolyl, isoindolyl, dihydrobenzothienyl, indolizinyl, cinnolinyl,phthalazinyl, quinazolinyl, naphthyridinyl, carbazolyl, benzodioxolyl,quinoxalinyl, purinyl, furazanyl, isobenzylfuranyl, benzimidazolyl,benzofuranyl, benzothienyl, quinolyl, indolyl, isoquinolyl, anddibenzofuranyl. “Heteroaryl” is unsubstituted or substituted with one tofive substituents independently selected from fluoro, hydroxy,trifluoromethyl, amino, C₁₋₄ alkyl, and C₁₋₄ alkoxy.

In some embodiments, the transaminases can be used in a process forpreparing a compound of structural formula (I):

having the indicated stereochemical configuration at the stereogeniccenter marked with an *; in an enantiomeric excess of at least 70% overthe opposite enantiomer, wherein

Z is OR² or NR²R³;

R¹ is C₁₋₈ alkyl, aryl, heteroaryl, aryl-C₁₋₂ alkyl, or heteroaryl-C₁₋₂alkyl;

R² and R³ are each independently hydrogen, C₁₋₈ alkyl, aryl, oraryl-C₁₋₂ alkyl; or

R² and R³ together with the nitrogen atom to which they are attachedform a 4- to 7-membered heterocyclic ring system optionally containingan additional heteroatom selected from O, S, NH, and NC₀₋₄ alkyl, theheterocyclic ring being unsubstituted or substituted with one to threesubstituents independently selected from oxo, hydroxy, halogen, C₁₋₄alkoxy, and C₁₋₄ alkyl, wherein alkyl and alkoxy are unsubstituted orsubstituted with one to five fluorines; and the heterocyclic ring systembeing optionally fused with a 5- to 6-membered saturated or aromaticcarbocyclic ring system or a 5- to 6-membered saturated or aromaticheterocyclic ring system containing one to two heteroatoms selected fromO, S, and NC₀₋₄ alkyl, the fused ring system being unsubstituted orsubstituted with one to two substituents selected from hydroxy, amino,fluorine, C₁₋₄ alkyl, C₁₋₄ alkoxy, and trifluoromethyl. In theseembodiments, the process comprises the step of contacting a prochiralketone of structural formula (II):

with an improved transaminase polypeptide disclosed herein in thepresence of an amino group donor in a suitable organic solvent undersuitable reaction conditions for the conversion of the compound offormula (II) to the compound of formula (I).

In some embodiments of the process, the R¹ of formula (II) is benzyl,wherein the phenyl group of benzyl is unsubstituted or substituted withone to three substituents selected from the group consisting offluorine, trifluoromethyl, and trifluoromethoxy.

In some embodiments of the process, the Z of formula (II) is NR²R³.

In some embodiments of the process, the NR²R³ of formula (II) is aheterocycle of the structural formula (III):

wherein R⁴ is hydrogen or C₁₋₄ alkyl which is unsubstituted orsubstituted with one to five fluorines.

In some embodiments, the transaminases can be used in a process forpreparing a compound of structural formula (1):

having the (R)-configuration at the stereogenic center marked with an***, in an enantiomeric excess of at least 70% over the enantiomerhaving the opposite (S)-configuration; wherein

Ar is phenyl which is unsubstituted or substituted with one to fivesubstituents independently selected from the group consisting offluorine, trifluoromethyl, and trifluoromethoxy; and

R⁴ is hydrogen or C₁₋₄ alkyl unsubstituted or substituted with one tofive fluorines. In such embodiments, the process comprises the step ofcontacting a prochiral ketone of structural formula (2):

with an improved transaminase polypeptide disclosed herein in thepresence of an amino group donor in a suitable organic solvent undersuitable reaction conditions for the conversion of the compound offormula (2) to the compound of formula (1).

In some embodiments of the process, the Ar of formula (2) is2,5-difluorophenyl or 2,4,5-trifluorophenyl, and R⁴ is trifluoromethyl.

In some embodiments of the process, the Ar of formula (2) is2,4,5-trifluorophenyl.

In some embodiments, the transaminases can be used in a process forpreparing a compound of formula (1a),(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine,in enantiomeric excess:

In these embodiments, the process comprises the step of contacting aprochiral ketone of structural formula (2a),4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one):

with an improved transaminase polypeptide disclosed herein in thepresence of an amino group donor in a suitable organic solvent undersuitable reaction conditions for the conversion of the compound offormula (2a) to the compound of formula (1a).

In some embodiments of the processes above, the compound of formula (I),the compound of formula (1) or the compound of formula (1a) is producedin at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or more enantiomeric excess.

In some embodiments of the processes, the compound of formula (I), thecompound of formula (1) or the compound of formula (1a) is produced inat least 99% enantiomeric excess.

In some embodiments of the process, the improved transaminases areselected from SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98,100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126,128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154,156, 158, 160, 162, 164, 166 or 168.

The compound of formula (II), the compound of formula (2), and thecompound of formula (2a), along with their synthesis, are described in,among others, U.S. Pat. No. 7,326,708 and 7,468,459, the disclosures ofwhich are incorporated herein by reference in their entirety.

As noted above, the transaminase polypeptide herein uses pyridoxalphosphate (PLP) as a coenzyme, which may be bound to the enzyme whenprepared, e.g., as provided by the host cell in which the polypeptide isexpressed. In some embodiments, PLP, PLP analogs, or precursors to PLPcan be added to the media of host cells during expression of thetransaminase polypeptide. In some embodiments of the processes, PLP orPLP analogs can be added to a reaction to provide the coenzyme requiredfor enzyme activity. The amount of PLP sufficient for enzyme activitycan be determined by one of skill in the art.

In some embodiments, the process comprises contacting or incubating theketoamide substrate4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-onewith an improved transaminase in presence of an amino group donor undersuitable reaction conditions to convert the ketoamide substrate to theproduct (2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-aminewith 50 to 100 times or greater conversion rate and/or activity thanthat of SEQ ID NO:4. Exemplary polypeptides comprise an amino acidsequence corresponding to SEQ ID NO: 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130,132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158,160, 162, 164, 166 or 168.

In some embodiments, the process comprises contacting or incubating theketoamide substrate4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-onewith an improved transaminase in presence of an amino group donor undersuitable reaction conditions to convert the ketoamide substrate to theproduct(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-aminewith 1.1 to 5 times or greater conversion rate and/or activity than thatof SEQ ID NO:22. Exemplary polypeptides comprise an amino acid sequencecorresponding to SEQ ID NO: 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84,86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168.

In some embodiments, the process comprises contacting or incubating theketoamide substrate4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-onewith an improved transaminase in presence of an amino group donor undersuitable reaction conditions to convert the ketoamide substrate to theproduct(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-aminewith 1.1 to 5 times or greater conversion rate and/or activity than thatof SEQ ID NO:48. Exemplary polypeptides comprise an amino acid sequencecorresponding to SEQ ID NO: 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,162, 164, 166 or 168.

In some embodiments, the process comprises contacting or incubating theketoamide substrate4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-onewith an improved transaminase in presence of an amino group donor undersuitable reaction conditions to convert the ketoamide substrate to theproduct(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-aminewith 1.1 to 5 times or greater conversion rate and/or activity than thatof SEQ ID NO:58. Exemplary polypeptides comprise an amino acid sequencecorresponding to SEQ ID NO: 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118,120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,148, 150, 152, 154, 156, 158, 160, 162, 164, 166 or 168.

In some embodiments of the processes above, the reaction condition forcarrying out the process can comprise a pH of about 7.0 to about 9.0. Insome embodiments, the reaction condition for the process is a pH ofabout 8.5.

In some embodiments, the reaction condition for carrying out the processcan comprise a temperature of about 25° C. to about 50° C. In someembodiments, the reaction condition is a temperature of about 45° C.

In some embodiments, the reaction condition is a pH of about 8.5 and atemperature of about 45° C.

In some embodiments of the process, the organic solvent comprises apolar solvent, such as methanol or DMSO.

In some embodiments, the organic solvent is DMSO, which can be presentfrom about 10% to about 40% volume/volume (v/v); about 25% to about 40%(v/v); 10% to about 50% (v/v) or about 25% to about 50% (v/v) of DMSO.In some embodiments, the DMSO is present at about 30% v/v, 35% v/v, 40%v/v, 45% v/v or about 50% v/v.

As discussed above, the amino group donor used in the process can be achiral amine or an achiral amine An achiral amino group donor has theadvantage of not being limited in its reaction to a specificstereoisomer, thus requiring less of the amino group donor. Varioussuitable amino group donors can be used, including, by way of exampleand not limitation, isopropylamine (also referred to as 2-aminopropane),L, D or DL alanine, phenylalanine, glutamate, glutamine, leucine (or anyother suitable α-amino acids), 3-aminobutyric acid (or any othersuitable β-amino acids), and methylbenzylamine. In some embodiments, theamino group donor is isopropylamine. In some embodiments, other aminogroup donors may be used, including, among others, α-phenethylamine(also termed 1-phenylethanamine), and its enantiomers(S)-1-phenylethanamine and (R)-1-phenylethanamine,2-amino-4-phenylbutane, glycine, L-glutamic acid, L-glutamate,monosodium glutamate, L-aspartic acid, L-lysine, L-ornithine, β-alanine,taurine, n-octylamine, cyclohexylamine, 1,4-butanediamine,1,6-hexanediamine, 6-aminohexanoic acid, 4-aminobutyric acid, tyramine,and benzyl amine, 2-aminobutane, 2-amino-1-butanol,1-amino-l-phenylethane, 1-amino-1-(2-methoxy-5-fluorophenyl)ethane,1-amino-1-phenylpropane, 1-amino-1-(4-hydroxyphenyl)propane,1-amino-1-(4-bromophenyl)propane, 1-amino-1-(4-nitrophenyl)propane,1-phenyl-2-aminopropane, 1-(3-trifluoromethylphenyl)-2-aminopropane,2-aminopropanol, 1-amino-1-phenylbutane, 1-phenyl-2-aminobutane,1-(2,5-dimethoxy-4-methylphenyl)-2-aminobutane, 1-phenyl-3-aminobutane,1-(4-hydroxyphenyl)-3-aminobutane, 1-amino-2-methylcyclopentane,1-amino-3-methylcyclopentane, 1-amino-2-methylcyclohexane,1-amino-1-(2-naphthyl)ethane, 3-methylcyclopentylamine,2-methylcyclopentylamine, 2-ethylcyclopentylamine,2-methylcyclohexylamine, 3-methylcyclohexylamine, 1-aminotetralin,2-aminotetralin, 2-amino-5-methoxytetralin, and 1-aminoindan, includingboth (R) and (S) single isomers where possible and including allpossible salts of the amines

In some embodiments of the processes above, the step in the process canfurther comprise removal of the carbonyl by-product formed from theamino group donor when the amino group is transferred to the amino groupacceptor. Such removal in situ can reduce the rate of the reversereaction such that the forward reaction dominates and more substrate isthen converted to product.

Removal of the carbonyl by-product can be carried in a number of ways.Where the amino group donor is an amino acid, such as alanine, thecarbonyl by product, a keto acid, can be removed by reaction with aperoxide (see, e.g., US 2008/0213845, incorporated herein by reference).Peroxides which can be used include, among others, hydrogen peroxide;peroxyacids (peracids) such as peracetic acid (CH₃CO₃H),trifluoroperacetic acid and metachloroperoxybenzoic acid; organicperoxides such as t-butyl peroxide ((CH₃)₃COOH), or other selectiveoxidants such as tetrapropylammonium perruthenate, MnO₂, KMnO₄,ruthenium tetroxide and related compounds. Alternatively, pyruvateremoval can be achieved via its reduction to lactate by employinglactate dehydrogenase to shift equilibrium to the product amine (see,e.g., Koszelewski et al., 2008, Adv. Syn. Catal. 350: 2761-2766).Pyruvate removal can also be achieved via its decarboxylation to carbondioxide acetaldehyde by employing pyruvate decarboxylase (see, e.g.,Höhne et al., 2008, Chem BioChem 9: 363-365).

In some embodiments, where the choice of the amino group donor resultsin a carbonyl by-product that has a vapor pressure higher than water(e.g., a low boiling co-product such as a volatile organic carbonylcompound), the carbonyl by-product can be removed by sparging thereaction solution with a non-reactive gas or by applying a vacuum tolower the reaction pressure and removing the carbonyl by-product presentin the gas phase. A non-reactive gas is any gas that does not react withthe reaction components. Various non-reactive gases include nitrogen andnoble gases (e.g., inert gases). In some embodiments, the non-reactivegas is nitrogen gas.

In some embodiments, the amino acid donor used in the process isisopropylamine, which forms the carbonyl by-product acetone upontransfer of the amino group to the amino group acceptor. The acetone canbe removed by sparging with nitrogen gas or applying a vacuum to thereaction solution and removing the acetone from the gas phase by anacetone trap, such as a condenser or other cold trap. Alternatively, theacetone can be removed by reduction to isopropanol using aketoreductase.

In some embodiments of the processes above where the carbonyl by-productis removed, the corresponding amino group donor can be added during thetransamination reaction to replenish the amino group donor and/ormaintain the pH of the reaction. Replenishing the amino group donor alsoshifts the equilibrium towards product formation, thereby increasing theconversion of substrate to product. Thus, in some embodiments whereinthe amino group donor is isopropylamine and the acetone product isremoved in situ, isopropylamine can be added to the solution toreplenish the amino group donor lost during the acetone removal and tomaintain the pH of the reaction (e.g., at about 8.5). Alternatively, inembodiments where an amino acid is used as amino group donor, the ketoacid carbonyl by-product can be recycled back to the amino acid byreaction with ammonia and NADH using an appropriate amino aciddehydrogenase enzyme, thereby replenishing the amino group donor.

In some embodiments, the process for converting ketoamide substrate4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-oneto product(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-aminecomprises contacting the ketoamide substrate at about 10 to 50 g/L withabout 1 to 20 g/L of a transaminase described herein under reactionconditions of pH 7.5 to 9.0 and a temperature of 30 to 50° C. inpresence of isopropylamine of from about 1 M to about 2 M, wherein atleast 80%, 85%, 90%, 92%, 94%, 96%, or 98% or more of the ketoamidesubstrate is converted to product in 24 hrs. In some embodiments, thetransaminase polypeptide capable of carrying out the foregoing reactioncomprises an amino acid sequence corresponding to SEQ ID NO: 80, 86, 96,98, 100, 102, 110, or 166.

In some embodiments, the processes above can further comprise the stepof isolating the compound of structural formula (I), the compound ofstructural formula (1), or the compound of structural formula (1a) fromthe reaction solvent.

In some embodiments, the processes above can further comprise a step ofconverting the compound of structural formula (1) or the compound ofstructural formula (1a) into a pharmaceutically acceptable salt bycontacting the compound with a pharmaceutically acceptable acid in asuitable reaction solvent. In some embodiments, the pharmaceuticallyacceptable acid is phosphoric acid and the pharmaceutically acceptablesalt is the dihydrogenphosphate salt. In some embodiments, the salt of(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amineis the phosphate monohydrate salt, having the following chemicalformula:

In some embodiments, in a process for the preparation of(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-aminephosphate (1:1) monohydrate, the improvement in the process comprises astep of converting the compound of formula (1a) to the compound offormula (2a) with a transaminase polypeptide of the disclosure inpresence of an amino group donor in a suitable organic solvent undersuitable reaction conditions, wherein the compound of formula (1a) is

and the compound of formula (2a) is:

In some embodiments of the preparation of the phosphate monohydratesalt, the amino donor is isopropylamine.

Methods for preparing various salts are described in U.S. Pat. No.7,326,708 and 7,468,459, each of which is hereby incorporated byreference herein. An exemplary process for preparing the phosphatemonohydrate of sitagliptin is presented in Example 13.

In some embodiments, the processes above can further comprise a step ofcrystallizing the pharmaceutically acceptable salt from the reactionsolvent.

Also provided herein are compositions of the transaminases andsubstrates/products. In some embodiments, the compositions can comprisethe compound of formula (I), the compound of formula (1) or the compoundof formula (1a) and an improved transaminase of the disclosure. Any oneor more of the improved engineered transaminases can be part of thecomposition.

In some embodiments, the composition can comprise the compound offormula (II), the compound of formula (2), or the compound of formula(2a) an improved transaminase described herein.

In some embodiments, the compositions can further comprise an aminogroup donor, e.g., of formula (3). In some embodiments of thecompositions, the amino group donor can comprise isopropylamine,alanine, 3-aminobutyric acid, or methylbenzylamine. In some embodimentsof the compositions, the amino group donor is an isopropylamine.

7. EXAMPLES

Various features and embodiments of the disclosure are illustrated inthe following representative examples, which are intended to beillustrative, and not limiting.

Example 1 Wild-Type Transaminase Gene Acquisition and Construction ofExpression Vectors

Transaminase (TA) encoding genes were designed for expression in E.coli. based on the reported amino acid sequence of the transaminase, anda codon optimization algorithm as described in Example 1 of USapplication publication 20080248539, which is hereby incorporated byreference herein. Genes were synthesized using oligonucleotides,generally composed of 42 nucleotides, and the gene cloned into theexpression vector pCK110700 (depicted as FIG. 1 in US applicationpublication 20050153417, which is hereby incorporated by referenceherein) or pCK110900 (depicted as FIG. 3 in US application publication20060195947, which is hereby incorporated by reference herein) under thecontrol of a lac promoter. This expression vector also contains the P15aorigin of replication and the chloramphenicol resistance gene. Resultingplasmids were transformed into E. coli. W3110 using standard methods.Codon optimized genes and the encoded polypeptides are listed in Table2, and their sequences provided as SEQ ID NOs:1 and 2.

Likewise, the genes encoding the engineered transaminases of the presentdisclosure listed in Table 2 (SEQ ID NOs: 3-168) were cloned into vectorpCK110700 or pCK110900 for expression in E. coli W3110.

Example 2 Production of Transaminase Powders—Shake Flask Procedure

A single microbial colony of E. coli containing a plasmid encoding atransaminase of interest was inoculated into 50 mL Luria Bertani brothcontaining 30 μg/mL chloramphenicol and 1% glucose. Cells were grownovernight (at least 16 hrs) in an incubator at 30° C. with shaking at250 rpm. The culture was diluted into 250 mL M9YE (1.0 g/L ammoniumchloride, 0.5 g/L of sodium chloride, 6.0 g/L of disodium monohydrogenphosphate, 3.0 g/L of potassium dihydrogen phosphate, 2.0 g/L ofTastone-154 yeast extract, 1 L/L de-ionized water) containing 30 μg/mLchloramphenicol and 100 μM pyridoxine, in a 1 liter flask to an opticaldensity at 600 nm (OD600) of 0.2 and allowed to grow at 30° C.Expression of the transaminase gene was induced by addition of isopropylβ D-thiogalactoside (IPTG) to a final concentration of 1 mM when theOD600 of the culture is 0.6 to 0.8 and incubation was then continuedovernight (at least 16 hrs). Cells were harvested by centrifugation(5000 rpm, 15 min, 4° C.) and the supernatant discarded. The cell pelletwas resuspended with an equal volume of cold (4° C.) 100 mMtriethanolamine (chloride) buffer, pH 7.5 containing 100 or 500 μMpyridoxal 5′-phosphate (PLP), and harvested by centrifugation as above.The washed cells were resuspended in two volumes of the coldtriethanolamine (chloride) buffer containing PLP and passed through aFrench Press twice at 12,000 psi while maintained at 4° C. Cell debriswas removed by centrifugation (9000 rpm, 45 min., 4° C.). The clearlysate supernatant was collected and stored at −20° C. Lyophilization offrozen clear lysate provides a dry powder of crude transaminase enzyme.Alternatively, the cell pellet (before or after washing) may be storedat 4° C. or 80° C.

Example 3 Production of Transaminase—Fermentation Procedure

A single microbial colony of E. coli. containing a plasmid with thetransaminase gene of interest was inoculated into 2 mL M9YE broth (1.0g/L ammonium chloride, 0.5 g/L of sodium chloride, 6.0 g/L of disodiummonohydrogen phosphate, 3.0 g/L of potassium dihydrogen phosphate, 2.0g/L of Tastone-154 yeast extract, 1 L/L de-ionized water) containing 30μg/ml chloramphenicol and 1% glucose. Cells were grown overnight (atleast 12 hrs) in an incubator at 37° C. with shaking at 250 rpm. Afterovernight growth, 0.5 mL of this culture was diluted into 250 ml M9YEBroth, containing 30 μg/ml chloramphenicol and 1% glucose in 1literflask and allowed to grow at 37° C. with shaking at 250 rpm. When theOD600 of the culture is 0.5 to 1.0, the cells were removed from theincubator and either used immediately, or stored at 4° C.

Bench-scale fermentations were carried out at 30° C. in an aerated,agitated 15 L fermentor using 6.0 L of growth medium (0.88 g/L ammoniumsulfate, 0.98 g/L of sodium citrate; 12.5 g/L of dipotassium hydrogenphosphate trihydrate, 6.25g/L of potassium dihydrogen phosphate, 3.3g/Lof Tastone-154 yeast extract, 0.083 g/L ferric ammonium citrate, and 8.3ml/L of a trace element solution containing 2 g/L of calcium chloridedihydrate, 2.2 g/L of zinc sulfate heptahydrate, 0.5 g/L manganesesulfate monohydrate, 1 g/L cuprous sulfate heptahydrate, 0.1 g/Lammonium molybdate tetrahydrate and 0.02 g/L sodium tetraborate. Thevessel was sterilized at 121° C. and 15 PSI for 30 minutes, and 100 μMpyridoxine was added post sterilization. The fermentor was inoculatedwith a late exponential culture of E. coli W3110 containing a plasmidencoding the transaminase gene of interest (grown in a shake flask asdescribed above to a starting OD₆₀₀ of 0.5 to 1.0. The fermentor wasagitated at 250-1250 rpm and air was supplied to the fermentation vesselat 0.6-25 L/min to maintain a dissolved oxygen level of 50% saturationor greater. The pH of the culture was maintained at 7.0 by addition of20% v/v ammonium hydroxide. Growth of the culture was maintained byaddition of a feed solution containing 500 g/L Cerelose dextrose, 12 g/Lammonium chloride and 5.1 g/L magnesium sulfate heptahydrate. After theculture reached an OD₆₀₀ of 70+−10, expression of transaminase wasinduced by addition of isopropyl-β-D-thiogalactoside (IPTG) to a finalconcentration of 1 mM and fermentation is continued for another 18hours. The culture was then chilled to 4° C. and maintained at thattemperature until harvested. Cells were collected by centrifugation at5000 G for 40 minutes in a Sorval RC12BP centrifuge at 4° C. Harvestedcells were used directly in the following downstream recovery process orthey may be stored at 4° C. or frozen at −80° C. until such use.

The cell pellet was resuspended in 2 volumes of 100 mM triethanolamine(chloride) buffer, pH 7.5 containing 100 or 500 μM pyridoxal5′-phosphate (PLP), at 4° C. to each volume of wet cell paste. Theintracellular transaminase was released from the cells by passing thesuspension through a homogenizer fitted with a two-stage homogenizingvalve assembly using a pressure of 12000 psig. The cell homogenate wascooled to −20° C. immediately after disruption. A solution of 11% w/vpolyethyleneimine pH 7.2 was added to the lysate to a finalconcentration of 0.5% w/v. A solution of 1 M Na₂SO₄ was added to thelysate to a final concentration of 100 mM. The lysate was then stirredfor 30 minutes. The resulting suspension was clarified by centrifugationat 5000 G in a Sorval RC12BP centrifuge at 4° C. for 30 minutes. Theclear supernatant was decanted and concentrated ten-fold using acellulose ultrafiltration membrane with a molecular weight cut off of 30kD. The final concentrate was dispensed into shallow containers, frozenat −20° C. and lyophilized to powder. The transaminase powder was storedat −80° C.

Example 4 High-Throughput Screening for Identification of Variants ofthe Arthrobacter sp. KNK168 Transaminase Capable of StereoselectivelyConverting Ketoamide Substrate to Sitagliptin

Achiral HPLC method to determine conversion of ketoamide substrate tositagliptin: Enzymatic conversion of ketoamide substrate (prepared asdescribed in U.S. Pat. No. 7,326,708) to sitagliptin was determinedusing an Agilent 1200 HPLC equipped with an Agilent Eclipse XDB-C8column (4.6×150 mm, 5 μm), using 45:55 10 mM NH₄Ac/MeCN as eluent at aflow rate of 1.5 ml/min and a column temperature 40° C. Retention times:ketoamide substrate: 1.7 min; sitagliptin: 1.4 min. The ketoamidesubstrate and product in the eluant were determined as the peak area at210 nm or 286 nm, with a path length of 1 cm. Using these conditions,the limit of detection for sitagliptin was 5 μg/mL. Generally, anincident wavelength of 210 nm was used for activity measurements fortransaminases with activity similar or equal to SEQ ID NO:4.

Chiral HPLC method to determine stereopurity of sitagliptin:Stereoisomeric purity of sitagliptin was determined using an Agilent1200 HPLC equipped with a Daicel Chiralpak AD-H column (4.6×150 mm, 5μm) using 60:40:0.1:0.1 EtOH/Heptane/diethylamine/water as the eluent ata flow rate of 0.8 ml/min and a column temperature of 35° C. Retentiontimes: ketoamide substrate: 6.3 min; (S)-enantiomer: 8.4 min;sitagliptin: 10.8 min. The ketoamide substrate and product weredetermined as the peak area at 210 nm or 268 nm with a path length of 1cm.

Liquid chromatography-mass spectroscopy (LC/MS) method for detectinglow-level conversion of ketoamide substrate to sitagliptin: Low-levelenzymatic conversion of ketoamide substrate to sitagliptin wasdetermined using an LC/MS/MS method. Five microliters of sample wasloaded into an Eclipse XDB-C8 HPLC column (4.6×150 mm) and elutedisocratically with a 40:60 mobile phase of 0.2% ammonium formate andmethanol at 1.0 mL/min. The retention time of sitagliptin was 1.5minutes at 35° C. Mass spectrometry was used for detection on a WatersQuattro triple quadruple. Q1 was set to pass the M+H ion at 408.1 AMUand Q3 was set to pass the 235.1 daughter ion. The collision cell (Q2)had a collision energy of 17.0 and Argon gas flow of 0.3 mL/min.Ionization was by APCI with a corona discharge of 5 μA, sourcetemperature of 130° C. and probe temperature of 600° C. Desolvation gasflow was 100 L/minute and the cone gas was set to 50 L/minute. Usingthese conditions the limit of detection for sitagliptin was 71 pg/mL.

Example 5 High-Throughput Screening for Identification of Variants ofthe Arthrobacter sp. KNK168 Transaminase Capable of StereoselectivelyConverting Ketoamide Substrate to Sitagliptin

The gene encoding transaminase, constructed as described in Example 1,was mutagenized using methods described above and the population ofaltered DNA molecules was used to transform a suitable E. coli hoststrain. Antibiotic resistant transformants were selected and processedto identify those expressing a transaminase with an improved ability totransaminate the ketoamide substrate stereoselectively to sitagliptin inthe presence of a suitable amino group donor (i.e., isopropylamine) Cellselection, growth, induced expression of transaminase variant enzymesand collection of cell pellets were as described below.

Recombinant E. coli colonies carrying a gene encoding transaminase werepicked using a Q-Bot® robotic colony picker (Genetix USA, Inc., Boston,Mass.) into 96-well shallow well microtiter plates containing in eachwell 180 μL LB Broth, 1% glucose and 30 μg/mL chloramphenicol (CAM).Cells were grown overnight at 30° C. with shaking at 200 rpm. A 10 μLaliquot of this culture was then transferred into 96-deep well platescontaining 390 μL M9YE broth, 100 μM pyridoxine and 30 μg/mL CAM. Afterincubation of the deep-well plates at 30° C. with shaking at 250 rpm for2-3 hrs, recombinant gene expression within the cultured cells wasinduced by addition of IPTG to a final concentration of 1 mM. The plateswere then incubated at 30° C. with shaking at 250 rpm for 18 hrs.

Cells were pelleted by centrifugation (4000 RPM, 10 min, 4° C.),resuspended in 200 μL lysis buffer and lysed by shaking at roomtemperature for 2 hours. The lysis buffer contained 100 mMtriethanolamine (chloride) buffer, pH 7.5 or 8.5, 1 mg/mL lysozyme, 500μg/mL polymixin B sulfate (PMBS) and 100 to 4000 μM PLP. After sealingthe plates with aluminum/polypropylene laminate heat seal tape (Velocity11, Menlo Park, Calif., Cat# 06643-001), they were shaken vigorously for2 hours at room temperature. Cell debris was pelleted by centrifugation(4000 RPM, 10 min., 4° C.) and the clear supernatant assayed directly orstored at 4° C. until use.

For screening in methanol or DMSO at pH 7.5, with early-stage engineeredtransaminases (i.e., early-stage “evolvants”), a 10 μL aliquot of asolution of ketoamide substrate (40 mg/mL) in methanol or DMSO was addedto each well of a Costar® deep well plate, followed by addition of 90 μLof 1.1 M isopropylamine hydrochloride using a Biomek NXp roboticinstrument (Beckman Coulter, Fullerton, Calif.). This was then followedby addition of 100 μL of the recovered lysate supernatant, alsoperformed using the Biomek NXp, to provide a reaction comprising of 2mg/ml ketoamide substrate, 500 mM isopropyl amine hydrochloride, 50 mMtriethanolamine pH 7.5, and 5% methanol or DMSO (v/v). The plates wereheat-sealed with aluminum/polypropylene laminate heat seal tape(Velocity 11, Menlo Park, Calif., Cat# 06643-001) at 175° C. for 2.5seconds and then shaken overnight (at least 16 hours) at 30° C.Reactions were quenched by the addition of 1 ml acetonitrile using aPhoenix Liquid Handling System (Art Robbins Instruments, Sunnyvale,Calif.). Plates were resealed, shaken for 5 min, and then centrifuged at4000 rpm for 10 min. A 200 μL aliquot of the cleared reaction mixturewas transferred to a new shallow well polypropylene plate (Costar#3365), sealed and analyzed as described in Example 4.

For screening in 25% DMSO at pH 8.5 with late-stage engineeredtransaminases (i.e., late-stage “evolvants”), a 50 μL aliquot of asolution of ketoamide substrate (400 mg/mL) in dimethyl sulfoxide (DMSO)was added to each well of a Costar® deep well plate, followed byaddition of 50 μL of 4 M isopropylamine hydrochloride using a Biomek NXrobotic instrument (Beckman Coulter, Fullerton, Calif.). This was thenfollowed by addition of 100 μL of the recovered lysate supernatant, alsoperformed using the Biomek NX, to provide a reaction comprising of 100mg/ml ketoamide substrate, 1 M isopropyl amine hydrochloride, 50 mMtriethanolamine pH 8.5, and 25% DMSO (v/v). The plates were heat-sealedwith aluminum/polypropylene laminate heat seal tape (Velocity 11, MenloPark, Calif., Cat# 06643-001) at 175° C. for 2.5 seconds and then shakenovernight (at least 16 hours) at 45° C. Reactions were quenched by theaddition of 1 ml acetonitrile using a Phoenix Liquid Handling System(Art Robbins Instruments, Sunnyvale, Calif.). Plates were resealed,shaken for 5 min, and then centrifuged at 4000 rpm for 10 min. A 10 μLaliquot of the cleared reaction mixture was transferred to a new shallowwell polypropylene plate (Costar #3365) containing 190 μL acetonitrile,sealed and analyzed as described in Example 4.

For screening in 50% DMSO at pH 8.5 with late-stage engineeredtransaminases (i.e., late-stage “evolvants”), a 100 μL aliquot of asolution of ketoamide substrate (100 mg/mL) in dimethyl sulfoxide (DMSO)was added to each well of a Costar® deep well plate, followed byaddition of 50 μL of 4 M isopropylamine hydrochloride using a Biomek NXrobotic instrument (Beckman Coulter, Fullerton, Calif.). This was thenfollowed by addition of 50 μL of the recovered lysate supernatant, alsoperformed using the Biomek NX, to provide a reaction comprising of 50mg/ml ketoamide substrate, 1 M isopropyl amine hydrochloride, 50 mMtriethanolamine pH 8.5, and 50% DMSO (v/v). The plates were heat-sealedwith aluminum/polypropylene laminate heat seal tape (Velocity 11, MenloPark, Calif., Cat# 06643-001) at 175° C. for 2.5 seconds and then shakenovernight (at least 16 hours) at 45° C. Reactions were quenched by theaddition of 1 ml acetonitrile using a Phoenix Liquid Handling System(Art Robbins Instruments, Sunnyvale, Calif.). Plates were resealed,shaken for 5 min, and then centrifuged at 4000 rpm for 10 min. A 10 μLaliquot of the cleared reaction mixture was transferred to a new shallowwell polypropylene plate (Costar #3365) containing 190 μL acetonitrile,sealed and analyzed as described in Example 4.

The transaminase of SEQ ID NO:2, expressed as in Examples 1 and 2exhibited no detectable activity on the ketoamide substrate using thedetection methods of Example 4. Variants of the Arthrobacter sp. KNK168transaminase capable of converting ketoamide substrate to sitagliptinwere identified using the approaches and procedures disclosed above.Multiple iterations of these processes, in which one or more improvedisolates from one round were used as the starting material forsubsequent rounds of mutagenesis and screening, were used to develop or“evolve” Arthrobacter sp. KNK168 transaminase variants with an improvedability to reduce ketoamide substrate stereoselectively to sitagliptin.

Example 6 Stereoselective Transamination in Methanol of KetoamideSubstrate by Engineered Transaminases Designated “+” in Table 2 Derivedfrom Arthrobacter sp. KNK168 Transaminase

Improved transaminases designated “+” in Table 2 derived fromArthrobacter sp. KNK168 transaminase were evaluated at preparative scalein DMSO as follows. A 500 μL solution of transaminase variant (20 mg/mL)in 100 mM triethanolamine-chloride buffer pH 7.5 with 250 μM pyridoxal5′-phosphate was added to 5 mL reaction vial equipped with a magneticstir bar. Subsequently, 450 μL of 1.1 M isopropylamine hydrochloride,followed by 50 μL of a solution of ketoamide substrate (40 mg/mL) inDMSO was added to the transaminase solution. The reaction was stirred at22° C. and monitored by HPLC analysis of samples taken periodically fromthe reaction mixture (see Example 4 for analytical conditions). Table 2provides the SEQ ID NO. corresponding to transaminase variantsdesignated “+”, the number of amino acid residue differences from thewild-type transaminase, and activity of each toward ketoamide substraterelative to that of the enzyme having the amino acid sequence of SEQ IDNO: 4.

For many engineered transaminases, conversion of ketoamide substrate tositagliptin can also be achieved using amino group donors such asD-alanine, 3-aminobutyric acid, or α-methylbenzylamine at a suitableconcentration.

Example 7 Stereoselective Transamination in Methanol of KetoamideSubstrate by Engineered Transaminases Designated “++” in Table 2 Derivedfrom Arthrobacter sp. KNK168

Improved transaminases designated “++” in Table 2 derived fromArthrobacter sp. KNK168 variants were evaluated at preparative scale inmethanol as follows. A 500 μL solution of transaminase variant (20mg/mL) in 100 mM triethanolamine-chloride buffer pH 7.5 with 250 μMpyridoxal 5′-phosphate was added to 5 mL reaction vial equipped with astir bar. Subsequently, 450 μL of 1.1 M isopropylamine hydrochloride,followed by 50 μL of a solution of ketoamide substrate (40 mg/mL) inmethanol was added to the transaminase solution. The reaction wasstirred at 22° C. and monitored by HPLC analysis of samples takenperiodically from the reaction mixture (see Example 4 for analyticalconditions). Table 2 provides the SEQ ID NO. corresponding totransaminase variants designated “++”, the number of amino acid residuedifferences from the wild-type transaminase, and activity of each towardketoamide substrate relative to that of the enzyme having the amino acidsequence of SEQ ID NO: 4.

Example 8 Stereoselective Transamination in Methanol of KetoamideSubstrate by Engineered Transaminases Designated “+++” in Table 2Derived from Arthrobacter sp. KNK168

Improved transaminases designated “+++” in Table 2 derived fromArthrobacter sp. KNK168 variants were evaluated at preparative scale inmethanol as follows. A 500 μL solution of transaminase variant (20mg/mL) in 100 mM triethanolamine-chloride buffer pH 7.5 with 250 μMpyridoxal 5′-phosphate was added to 5 mL reaction vial equipped with astir bar. Subsequently, 450 μL of 2.2 M isopropylamine hydrochloride,followed by 50 μL of a solution of ketoamide substrate (100 or 200mg/mL) in methanol was added to the transaminase solution. The reactionwas stirred at 30° C. and monitored by HPLC analysis of samples takenperiodically from the reaction mixture (see Example 4 for analyticalconditions). Table 2 provides the SEQ ID NO. corresponding totransaminase variants designated “+++”, the number of amino acid residuedifferences from the wild-type transaminase, and activity of each towardketoamide substrate relative to that of the enzyme having the amino acidsequence of SEQ ID NO: 22.

Example 9 Stereoselective Transamination in Methanol of KetoamideSubstrate by Engineered Transaminases Designated “++++” in Table 2Derived from Arthrobacter sp. KNK168

Improved transaminases designated “++++” in Table 2 derived fromArthrobacter sp. KNK168 variants were evaluated at preparative scale inmethanol as follows. A 500 μL solution of transaminase variant (20mg/mL) in 100 mM triethanolamine-chloride buffer pH 8.5 with 250 μMpyridoxal 5′-phosphate was added to 5 mL reaction vial equipped with astir bar. Subsequently, 400 μL of 2.5 M isopropylamine hydrochloride,followed by 100 μL of a solution of ketoamide substrate (200 mg/mL) inmethanol was added to the transaminase solution. The reaction wasstirred at 45° C. and monitored by HPLC analysis of samples takenperiodically from the reaction mixture (see Example 4 for analyticalconditions). Table 2 provides the SEQ ID NO. corresponding totransaminase variants designated “++++”, the number of amino acidresidue differences from the wild-type transaminase, and activity ofeach toward ketoamide substrate relative to that of the enzyme havingthe amino acid sequence of SEQ ID NO: 48. Example 10

Stereoselective Transamination in DMSO of Ketoamide Substrate byEngineered Transaminases Designated “+++++” in Table 2 Derived fromArthrobacter sp. KNK168

Improved transaminases designated “+++++” in Table 2 derived fromArthrobacter sp. KNK168 variants were evaluated at preparative scale inDMSO as follows. A 250 μL solution of transaminase variant (20 mg/mL) in100 mM triethanolamine-chloride buffer pH 8.5 with 250 μM pyridoxal5′-phosphate was added to 5 mL reaction vial equipped with a stir bar.Subsequently, 500 μL of 2 M isopropylamine hydrochloride, followed by250 μL of a solution of ketoamide substrate (200 mg/mL) in DMSO wasadded to the transaminase solution. The reaction is stirred at 45° C.and monitored by HPLC analysis of samples taken periodically from thereaction mixture (see Example 4 for analytical conditions). Table 2provides the SEQ ID NO. corresponding to transaminase variantsdesignated “+++++”, the number of amino acid residue differences fromthe wild-type transaminase, and activity of each toward ketoamidesubstrate relative to that of the enzyme having the amino acid sequenceof SEQ ID NO: 58.

Example 11 Stereoselective Transamination in DMSO of Ketoamide Substrateby Engineered Transaminases Designated “++++++” in Table 2 derived fromArthrobacter sp. KNK168

Improved transaminases designated “++++++” in Table 2 derived fromArthrobacter sp. KNK168 variants were evaluated at preparative scale inDMSO as follows. A 250 μL solution of transaminase variant (8 mg/mL) in100 mM triethanolamine-chloride buffer pH 8.5 with 4000 μM pyridoxal5′-phosphate was added to 5 mL reaction vial equipped with a stir bar.Subsequently, 250 μL of 4 M isopropylamine hydrochloride, followed by500 μL of a solution of ketoamide substrate (100 mg/mL) in DMSO wasadded to the transaminase solution. The reaction is stirred at 45° C.and monitored by HPLC analysis of samples taken periodically from thereaction mixture (see Example 4 for analytical conditions). Table 2provides the SEQ ID NO. corresponding to transaminase variantsdesignated “++++++”, the number of amino acid residue differences fromthe wild-type transaminase, and activity of each toward ketoamidesubstrate relative to that of the enzyme having the amino acid sequenceof SEQ ID NO: 104.

Example 12 Process I for Conversion of Ketoamide Substrate toSitagliptin

The following example illustrates a large scale process used to increaseconversion of substrate4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-oneto product (2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,49triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine.This process uses nitrogen sparging to remove the acetone co-product andincrease the conversion of substrate to product. The addition ofisopropylamine in water helps keep the volume constant and maintain thepH of the reaction.

The large scale process contains the following reaction components:

Substrate Ketoamide: 20 g (98%) (48.2 nmole) Isopropylamine-HCl: 18.44 g(193 nmole) Pyridoxal phosphate PLP: 200 mg (98%) (0.79 nmole)Transaminase (SEQ ID NO: 86): 2.2 g 0.1M triethanolamine pH 8.5: 140 mLDMSO: 20 mL + 40 mL 4M iPr amine (free base): 38.5 mL

Process. To a 500 mL three neck round bottom (RB) flask with 4-bafflesand fitted with stirrer, pH probe, temp. probe, needle for nitrogensparge, and vacuum needle was added 18.25 g of isopropylaminehydrochloride (4 equiv.) followed by 200 mg of pyridoxal 5′-phosphatehydrate (vitamin B6). This was dissolved in 140 mL of pH 8.5, 0.1 Mtriethanolamine buffered water. DMSO (20 ml) was added followed by 2 gof transaminase enzyme (SEQ ID NO: 86) powder. The solution was broughtto 45° C. and the pH was again adjusted to 8.5 with 4 M aqueousisopropylamine. After this stabilized (˜5 min.), a solution of 20 gsubstrate dissolved in 40 mL of DMSO was added over 3 h. During theaddition, and throughout the reaction, pH steadily dropped. The pH wascontrolled by continuous addition of 4 M aqueous isopropylamine when pHdecreased by more than 0.1 units. In addition, nitrogen was spargedthrough the reaction after 2 h until 12 h. After 21 h, the conversionwas at 93%. A total of 38.5 mL of 4M isopropyl amine (3.056 equiv.) wasadded to the reaction during the process. The pH control unit had athermocouple, pH probe, and 4 M isopropylamine in water to control pH.

A substrate-product eneamine adduct impurity present in the productmixture (retention time of 4.1 min under the separation conditionsdescribed below) was destroyed by acidifying the mixture to pH 2.0 with10.5 mL 6 N HCl followed by stirring for 1 h at 45° C. Subsequently, 6 gCelite was added, stirred another hour, and then filtered through aCelite pad (85 mm ID frit, 10 mm thick pad of wetted Celite) withagitated washings (4×30 mL) of water/DMSO (90/10 with 1 drop 6 N HCl).Assays show a yield of 91% sitagliptin and 7% substrate ketoamide.

The product was further processed by adding 200 mL isopropyl acetate(IPac) followed by 32 mL 5 N NaOH with stirring until the pH was ˜11.Layers were separated (35 min. settling with emulsion in organic layer)and extracted aqueous layer with another 200 mL isopropyl acetate. Afinal extraction was carried out with an additional 100 mL of isopropylacetate. All three isopropyl acetate layers were combined and allowed tosettle for 30 min and the residual water drained off. The product in theorganic layer was then washed with 150 mL brine (settles in <1 h),separated and dried with Na₂SO₄, and then filtered. The solvent wasswitched to isopropanol (66.29 g isopropanol (IPA) solution). Assay ofthe final product shows the following:

26.8wt. % sitagliptin (17.73g).

1.89wt. % ketoamide substrate (1.25g).

˜60.3mL IPA

The HPLC conditions for separation of the product mixture were asfollows:

Column: Zorbax Eclipse Plus C18, 4.6×50 mm, 1.8 um

Gradient: min H₂O (0.1% H₃PO₄)/CH₃CN 0 90/10 5  5/95 6  5/95 6.01 90/108 stop

Flow: 1.5 mL/min

Column Temperature: 25° C.

Sample volume:5 μL

Detector: UV @ 210 nm

Samples for HPLC analysis were prepared in 0.2 mg/mL in 1/1H₂O(0.1%H₃PO₄)/CH₃CN. The retention times under the chromatographic conditionsabove were as follows:

Sitagliptin: 2.2 min

Ketoamide substrate: 3.2 min

Ketoamide substrate (enol): 3.9 min

Substrate-product eneamine: 4.1 min.

The purging with nitrogen gas removes acetone, the product of thetransamination reaction, thereby shifting the equilibrium of thetransaminase catalyzed reaction towards product formation, and hence ahigher percentage conversion of substrate to product. In addition, thecontinual addition of isopropylamine not only maintains the pH of thereaction condition, but also replenishes the amino group donor lost inthe transamination reaction. Although the transaminase polypeptidehaving SEQ ID NO: 86 was used in this process, it is to be understoodthat this exemplary process can employ any of the subsequent engineeredtransaminases disclosed herein.

Example 13 Process II for Increasing Conversion of Ketoamide Substrateto Sitagliptin

The following example illustrates a second large scale process used toincrease conversion of substrate4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-oneto product(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine.This process uses a vacuum to remove the acetone product and increasethe conversion of substrate to product. As in the preceding example,addition of isopropylamine in water helps keep the volume constant andmaintain the pH of the reaction.

Materials.

Mole Reaction component Amount Equivalents Ketoamide substrate: 1000 g(96%) (2.36 mole) 1.0 Isopropylamine-HCL: 903 g (9.45 mole) 4.0Pyridoxal phosphate PLP: 10 g (98%) (0.04 mole) 0.017 Transaminase 50.0g (SEQ ID NO: 102): Triethanolamine: 104 g DMSO: 1.5 L + 2 L 4M aqueousisopropylamine 157 mL (free base):

Equipment for the process has the following features: a pH control unitthat has thermocouple, a pH probe, and reservoir of 4M isopropylamine inwater to control pH. The reaction vessel is connected to a vacuum lineand corresponding controller (set to 375 ton) and ReactIR probe (MetterToledo, Maryland, USA) to measure acetone and product formation.

Process. A 22 L mechanically stirred round bottom (RB) flask was fittedwith a ReactIR probe, a base feed line connected to a reservoir of 4 Maqueous isopropylamine, a ketoamide feed line connected to a reservoirof ketoamide substrate, a pH probe, and a vacuum line to a control boxand a trap. The flask was charged with 900 g of isopropylaminehydrochloride followed by addition of 6.4 L deionized water and 93 mL oftriethanolamine (2 m/s tip speed) and 10 g pyridoxal 5′-phosphate (pH8.4) was added. This was followed by charging of the flask with 50 g ofdissolved transaminase polypeptide having SEQ ID NO: 102. After 10 min.of stirring at RT, 1.5 L of DMSO was added over 30 min, and the reactorwas warmed to 40° C. After the temperature stabilized, the pH probe wasexchanged for a temperature probe, and the pH maintained at pH 8.5 witha pH controller and 4 M isopropylamine solution. Ketoamide substrate (1Kg) was dissolved in 2 L of DMSO and placed into a 5 L addition funnel.Vacuum (˜500 torr initially then 375 torr overnight after addition) wasapplied to the reaction vessel, and the ketoamide solution was added tothe reactor over 4 hrs (667 mL/h). A total of 1.45 equiv. ofisopropylamine was added after 25 h. There was about 94% conversion ofsubstrate to product.

After allowing the reaction to proceed for 1 day, 580 mL 5 N HCl wasadded to the reaction solution until pH 2 and the solution stirred for 2hrs at 45° C. The solution was filtered through a wide diameter Buchnerfunnel using two layers of cotton towel resulting in 12.3 kg offiltrate. The filtrate residue was agitated in 5% DMSO in 0.01 N HCl andthen rinsed with an additional 3×2 L of 5% DMSO in 0.01 N HCl (6.6 kgtotal). The residue was placed in aqueous acidic solution, washed (˜18 Ltotal) into an extractor and then placed in 9 L of isopropyl acetate.The pH was adjusted to 10 with 5 N NaOH (1.4 L), the solution agitatedat 165 RPM in 50 L ChemGlass vessel #1, the layers allowed to settle forabout 10 min, and the isopropyl acetate separated out. The solution wasextracted again with 9 L of isopropyl acetate, and the extractedisopropyl acetate layers were combined and allowed to settle for 20 h.The isopropyl acetate layers were washed with 6 L of brine (5.9 kg). TheIPAc solution was assayed and contained 861 g sitagliptin. Solvent wasswitched from IPAc (861 g in 19 L IPAc, 90% assay yield) to IPA onrotary vacuum evaporator by feeding over 1 h at 30° C. and concentratingto 50% volume. At this point, the sitagliptin free base precipitated outof solution. 2 L of 1% water in IPA was added to dissolve theprecipitate. 8 L of 1% water in IPA over 1 h was added at 35-40 ° C.bath temperature. Since additional precipitates form, additional 400 mLwater was added to dissolve the precipitate. The solution was allowed tosit on a rotary vacuum evaporator overnight, and then transferred toanother round bottom flask along with an additional 2 L of 1% water inIPA. Concentration gave 2.5746 kg of an IPA/water solution ofsitagliptin.

Example 14 Preparation of Sitagliptin Phosphate Monohydrate

Preparation of sitagliptin phosphate monohydrate is illustrated asfollows:

Materials for the preparation of the phosphate monohydrate salt ofsitagliptin is as follows:

Mole Reaction component Amount Equivalents Crude 3 757 g (1.86 mole) 1.045% w/w H₃PO₄ 411 g (1.89 mole) 1.02 water 347 + 491 + 100 mL 0.017Isopropanol 1.63 + 1.36 + 0.17 + 0.5 + 0.5 + 1.65 L Seed 5 g 0.005 88/12isopropanol/water 1.4 L

Process. To a solution containing 757 g Crude 3 in 1630 mL isopropanoland 347 mL water was added 1.36 L isopropanol followed by 491 mL ofdeionized water. The solution was transferred to 20 L stirred vessel,and then charged with 411 g of 45% w/w H₃PO₄ (Fisher 85%) and 172 mLIPA. Solution was heated to 72-80° C. to dissolve initial phosphate saltand charged with an additional 100 mL of water and 500 mL isopropanol tocompletely dissolve the phosphate salt. Solution was cooled to 62-66° C.and seeded with 5 g of pure sitagliptin phosphate. Reaction was allowedto sit for 3 h at 60-65° C., and then cooled to 20-25° C. over 5 h andthen additionally overnight. The reaction was charged with 2.65 Lisopropanol over 2 h (solution is ˜6:1 isopropanol/water) and allowed tosit for 1 h at RT and 2 h at 2° C. The material was passed through afilter that was prepared by wetting with 88/12 isopropanol/water. Theresulting cake was washed with a total of 1.4 L 88/12 isopropanol/waterand dried under atmosphere for about 3 h, and then transferred to a trayto dry at ˜40° C. in vacuum oven with nitrogen sweep (200 torr) for 3days providing 966 g sitagliptin phosphate hydrate. The material met (orexceeded) all purity specifications for manufactured sitagliptinphosphate hydrate and showed no residual solvent, enzyme (<18 ppm), PLPco-factor (<0.1 ppm), or endotoxin (<0.05 ng).

Example 15 Process III for Increasing Conversion of Ketoamide Substrateto Sitagliptin

The following example illustrates a third large scale process used toincrease conversion of substrate4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-oneto product(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine.Generally, the process uses the same equipment and conditions asdescribed in Example 12 but with a higher concentration of DMSO andsubstrate.

Process: The reaction was run in a vessel fitted with a mechanicalstirrer, temperature probe, pH probe, and a base addition line. The baseaddition line was used to control pH between 8.6 and 8.4 using a feed of4 M isopropylamine free base in water. To the vessel was added 1.92 Lwater, followed by 109 mL (0.82 mol, 0.33 equiv.) triethanolamine and1.64 L (6.56 mol, 2.67 equiv.) of 4 M isopropylamine solution. The pHwas then adjusted to 8.5 using 12 N HC1 (424 mL). The reactor was thencharged with 6.7 g (0.027 mol, 0.011 equiv.) of PLP followed by 40 g ofthe transaminase polypeptide having SEQ ID NO: 110 and the mixture wascarefully dissolved with gentle agitation. The vessel was placed on thereactor block with the temperature probe, base addition line, pH probe,and stirrer set to 400 RPM (Note: pH control loop is off at this point).Next, 2.22 L of DMSO was added into the stirring solution and thereactor was heated to 45° C. When the temperature stabilized, the pHcontrol loop was turned on and adjusted to pH to 8.5 (pH controlled with4 M isopropylamine in water). At this point, stirring was increased to600 RPM, but tip speed is kept below 2 m/s to avoid vortexing. Then, 1.0kg (corrected weight is 1 kg as received ketoamide is typically 96-98 wt% as a hemi-hydrate; 2.46 mol, 1.00 equiv.) of ketoamide was dissolvedinto 1.11 L of DMSO. This DMSO/ketoamide solution was then added to thereactor over 2-3 h. The reactor was then stirred at 45° C. and with thepH maintained between 8.6-8.4 for another ˜13 h with acetone removalbeing accomplished with 300 ton vacuum and 2 fps nitrogen sweep. After˜15 h total reaction time (1.3-2.0 equiv. isopropylamine uptake), thereaction was at 90-95% conversion as judged by reverse phase HPLCanalysis.

As described below, either a filtration or a direct extraction work-upprocedure can be used to prepare the product for downstream processing.

Filtration work-up: The pH control loop was turned off and 13 g ofsolka-floc was added to the vessel followed by 12 M HCl until pH 2-3.The reaction was then aged 1-2 h at 45° C. and 1000 RPM. The slurry wasthen passed through a filter (e.g., fritted plastic Buchner with filterpaper on 1 kg scale, or sparkle filter with no recycle loop on pilotplant scale). The vessel and filter was rinsed with 1 L of 0.01 N HCl.To this aqueous acidic filtrate was then added 3 L of IPAc and the pH ofthe aqueous phase was then adjusted to pH 11 with 19 N NaOH. The layerswere agitated with stirring, and then allowed to settle and separated(mild heat or vacuum accelerates phase separation). This was repeatedtwice more with 3 L of IPAc and the combined organics were then washedwith 3 L of brine (at pH 11). The resulting IPAc solution of thesitagliptin free base was then assayed for yield (typically 88-92% assayyield; 882-922 g) and solvent switched to IPA for downstream processingto sitagliptin phosphate monohydrate.

Direct extraction work-up: The pH control loop was turned off and 12 MHCl was added until pH 2-3. The reaction was then aged 1-2 h at 45° C.and 1000 RPM. The batch was cooled to RT and then 3 L of IPA was added,followed by 3 L of IPAc. The pH of the aqueous layer was then adjustedto 11 with 19 N NaOH. The mixture was agitated at 20-45° C. (heat may beused to break the emulsion), and then allowed to settle and separate.The IPAc/IPA layer was set aside and the aqueous layer was extractedwith 3 L of 80/20 (vol/vol) IPAc/IPA. The combined IPAc/IPA extractswere then washed with 3 L of brine. The resulting IPAc/IPA solution ofthe sitagliptin free base was then assayed for yield (typically 87-90%assay yield, 872-902 g) and solvent switched to IPA for downstreamprocessing to sitagliptin phosphate monohydrate

Example 16 Process IV for Increasing Conversion of Ketoamide Substrateto Sitagliptin

The following example illustrates a fourth large scale process used toincrease conversion of the ketoamide substrate to the sitagliptinfreebase product and the subsequent preparation of the sitagliptinphosphate. Generally, the process uses the same equipment conditions asdescribed in Examples 12, 14, and 15 but with alterations as detailedbelow.

A buffer solution was prepared by combining 0.59 L 4M isopropyl aminesolution, 0.67 L water, and 39 mL triethanolamine at 0-35° C. The pH ofthe buffer was adjusted to 8.4-9.2 at 20-25° C. using 12N hydrochloricacid. To this mixture was charged 1.22 g PLP and 16.25 g of thetransaminase polypeptide of SEQ ID NO: 110 at 15-25° C. The PLP andenzyme were dissolved with agitation. Next, 0.72 L DMSO was charged tothe batch at 15-46° C. over a minimum of 30 minutes. The enzyme mixturewas then heated to 44-46° C. and then adjusted to pH 8.4-8.7 with 4Misopropyl amine solution. Until the enzyme mixture was quenchedfollowing reaction, the pH was monitored and 4M isopropyl amine solutionwas charged as necessary to maintain the pH within the range of 8.4-8.7.

The ketoamide substrate,4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one(406.28 g, 1.00 mole) was dissolved into 0.48 L of DMSO, and thisDMSO/ketoamide solution was added to the enzyme mixture over 2-8 hours.This enzyme mixture was then allowed to react at 44-46° C. and pH8.4-8.7 for another 7-22 hours. During both the DMSO/ketoamide solutionaddition and the subsequent reaction, the pressure of the reactor wasvaried as necessary to evaporate acetone and thereby push the reactionto formation of product sitagliptin free base. In addition, during thecourse of the reaction, 4M isopropyl amine solution was charged(typically a minimum of 0.25 L, or 1.0 molar equivalent) as necessary inorder to maintain the pH at 8.4-8.7.

The reaction mixture was quenched by charging 65 g solka floc at 44-46°C. Following the solka floc charge, 12N hydrochloric acid was charged tothe batch until pH 2-3 is reached. The mixture was stirred at 44-46° C.and pH 2-3 for at least 3 hours. The mixture was then filtered at 44-46°C., and the waste cake washed with 0.01N hydrochloric acid (1.02 L-4.47L). The combined filtrate and 0.01N hydrochloric acid wash was cooled to15-25° C., and then 2.44 L of isopropyl acetate (IPAc) was added. Theapparent pH was then adjusted to 10.5-11.5 with 19N sodium hydroxide.The mixture was then agitated at 15-40° C., settled, and separated. Thetop, organic layer was set aside and the lower, aqueous layer was thenextracted with 1.22 L of IPAc at 15-25° C. The two organic extracts werethen combined and washed with water (0.20 L -1.23 L) at 15-40° C. Thefinal organic layer yielded 4.07 L of a sitagliptin freebase crudestream at 100 g/L (407.31 g, 1.00 mole) that was used in the preparationof the sitagliptin phosphate monohydrate.

The 4.07 L of sitagliptin freebase crude stream was concentrated undervacuum to 1.68 L at 20-35° C. and then solvent switched to isopropanolusing a minimum of 2.52 L of isopropanol. To this solution was charged aminimum of 0.27 L water to dissolve all solids. The addition of waterenhances formation of the monohydrate at the start of thecrystallization. Aqueous phosphoric acid (1.02 mole) was added to thissolution, then heated to dissolve.

The solution then was cooled to 62-68° C., seeded with 2.62 g of milledsitagliptin phosphate monohydrate (e.g., mean volume of 10-20 micronswith 95^(th) percentile between 25-45 microns as determined by Microtracanalysis) and allowed to age for 3-6 hours at 62-68° C. The slurry wascooled to 20-25° C. over a minimum of 2 hours. To the slurry was charged0.48 L of isopropanol over a minimum of 2 hours, while maintainingtemperature at 20-25° C. The slurry was then cooled to −15° C. to 20° C.over a minimum of 2 hours. The slurry was then filtered at −15° C. to20° C. and the wetcake washed with aqueous isopropanol (minimum watercontent of 8 wt %). The wetcake was dried at a maximum temperature of45° C. in vacuo to yield sitagliptin phosphate monohydrate.

Example 17 Process V for Increasing Conversion of Ketoamide Substrate toSitagliptin

The following example illustrates a fifth large scale process used toincrease conversion of the ketoamide substrate to the sitagliptinfreebase product and the subsequent preparation of the sitagliptinphosphate. Generally, this large scale process uses the same equipmentand conditions as described in Example 16 but with some alterations asdetailed below.

Buffer solution was prepared by combining 50.68 L 4M isopropyl aminesolution, 58.1 L water, and 3.36 L triethanolamine at 20-25° C. The pHof the buffer was then adjusted to 8.8-9.2 at 20-25° C. using 12Nhydrochloric acid. To the batch mixture was charged 0.11 kg of PLP, andthen 1.40 kg of the transaminase enzyme of SEQ ID NO: 110 at 20-25° C.The PLP and enzyme were dissolved with agitation (confirmed after 30min). Next, 61.76 L of DMSO was charged to the batch at 20-46° C. Thebatch was then heated to 44-46° C. and once at temperature, 4M isopropylamine solution was charged to adjust the pH to 8.4-8.7.

The ketoamide substrate (35.00 kg) was dissolved into 41.18 L of DMSO,and this DMSO/ketoamide solution added to the batch over 2-3 hours. Thebatch was then allowed to react at 44-46° C. and pH 8.4-8.7 for another12-22 hours. During both the DMSO/ketoamide solution addition and thesubsequent aging, the pressure of the reactor was varied in order toremove acetone (typical pressure conditions for acetone removal:˜325-350 mm Hg vacuum and ˜3-6 scfm nitrogen headspace sweep). Inaddition, during the course of the reaction, 4M isopropyl amine solutionwas charged as necessary in order to maintain the pH at 8.4-8.7. Typicalend of reaction conversion results are 88-93% after 15-17 hours of totalreaction time, which includes the DMSO/ketoamide transfer time.

The reaction was quenched by charging 5.60 kg solka floc slurry in 42 Lwater. Following the solka floc charge, 12N hydrochloric acid wascharged to the batch until pH 2-3 was reached. The reaction was stirredat 44-46° C. and pH 2-3 for 3 hours. The batch was then filtered at44-46° C., and the waste cake washed with 154 L of 0.01N hydrochloricacid solution.

The combined filtrate and wash solution was cooled to 15-25° C., andthen 210 L of isopropyl acetate (IPAc) added. The apparent pH of thebatch was then adjusted to 10.5-11.5 with 19N sodium hydroxide. Themixture was then agitated at 15-25° C., settled, and separated(Extraction #1). The top, organic layer was set aside and the lower,aqueous layer was then extracted with 105 L of IPAc at 15-25° C.(Extraction #2). The two organic extracts were then combined and washedwith 17.50 L water at 32-38° C. (Extraction #3). The final organic layeryielded 29.44 assay kg of sitagliptin freebase crude stream (˜85.9%assay yield).

The freebase crude stream, 294.40 L at 100 g/L (29.44 kg, 72.28 mole),was concentrated under vacuum (30-60 mmHg) to 121.59 L at 20-35° C. Thebatch was solvent switched to isopropanol using 182.39 L of isopropanol.To the batch was charged 19.72 L of water to dissolve all solids. Then,15.87 kg of 45 wt % aqueous phosphoric acid was added to the batch,which was heated to 72-80° C. to dissolve. The batch solution was cooledto 62-66° C. and seeded with 0.19 kg pin milled sitagliptin phosphatemonohydrate. The batch was aged for 3 hours at 62-66° C. and then cooledto 20-25° C. over 2 hours. To the batch was charged 34.40 L ofisopropanol over 2 hours, while maintaining batch temperature at 20-25°C. The batch was then cooled to a −15° C. to 0° C. over 2 hours. Theslurry was then filtered at −15° C. to 0° C. and the wetcake washed with70.05 L aqueous isopropanol (minimum of 8 wt % water). The wetcake wasdried at 40° C. in vacuo to yield sitagliptin phosphate monohydrate(37.34 physical kg, ˜98% yield).

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention(s).

What is claimed is:
 1. A recombinant transaminase polypeptide comprisingan amino acid sequence at least 90% identical to SEQ ID NO:
 2. 2. Therecombinant transaminase polypeptide of claim 1, wherein the amino acidsequence of the recombinant transaminase polypeptide comprises a residuedifference as compared to SEQ ID NO: 2 at one or more residue positionsselected from: X8, X94, X136, and X209.
 3. The recombinant transaminasepolypeptide of claim 2, wherein the amino acid residue differences ofthe recombinant transaminase polypeptide are selected from thefollowing: X8 is proline; X94 is leucine, isoleucine, or valine; X136 isleucine; and X209 is leucine.
 4. The recombinant transaminasepolypeptide of claim 1, wherein said recombinant transaminasepolypeptide is capable of converting4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-one(“ketoamide substrate”) to(2R)-4-oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine(“product”) under a defined reaction condition in the presence of anamino group donor isopropylamine to levels of the product detectable byHPLC-UV at 210 nm, where the reaction condition comprises about 2 g/Lketoamide substrate, about 0.5 M isopropylamine, about 22° C., about pH7.5, about 5% DMSO, about 100 μM pyridoxal phosphate, and about 20 mg/mLof transaminase polypeptide.
 5. The recombinant transaminase polypeptideof claim 1, wherein said recombinant transaminase polypeptide is capableof converting the ketoamide substrate to the product with an activitythat is equal to or greater than the activity of the polypeptide of SEQID NO: 4 under the defined reaction condition.
 6. The recombinanttransaminase polypeptide of claim 1, wherein said recombinanttransaminase polypeptide is capable of converting the ketoamidesubstrate to product in at least 90% enantiomeric excess.
 7. Therecombinant transaminase polypeptide of claim 1, wherein saidrecombinant transaminase polypeptide is capable of converting theketoamide substrate to product in at least 99% enantiomeric excess.
 8. Arecombinant polynucleotide encoding the recombinant transaminasepolypeptide of claim
 1. 9. An expression vector comprising therecombinant polynucleotide of claim
 8. 10. The expression vector ofclaim 9, further comprising at least one control sequence.
 11. Theexpression vector of claim 10, wherein said control sequence comprises apromoter.
 12. The expression vector of claim 10, wherein said controlsequence comprises a secretion signal.
 13. A host cell comprising theexpression vector of claim
 9. 14. The host cell of claim 13, whereinsaid host cell is a bacterial cell.
 15. The host cell of claim 14,wherein said bacterial cell is E.coli.